Application of histidine-rich glycoprotein in predicting, preventing or treating hemorrhagic transformation after thrombolytic therapy in patients with cerebral infarction

Abstract

The present invention belongs to the field of medical technology, and specifically relates to the application of histidine-rich glycoproteins in predicting, preventing or treating hemorrhagic transformation after thrombolytic therapy in patients with cerebral infarction. Histidine-rich glycoproteins can be used as markers to predict outcomes related to hemorrhagic transformation after thrombolytic therapy in patients with cerebral infarction, and have important clinical application value for risk assessment of whether patients have hemorrhagic transformation after thrombolytic therapy for cerebral infarction. At the same time, histidine-rich glycoproteins can inhibit rtPA-induced neutrophil NETosis and NETs formation, proinflammatory cytokine release, ROS production, blood-brain barrier damage, and neutrophil infiltration of the central nervous system, and can effectively reduce intracerebral hemorrhagic transformation caused by delayed administration of rtPA and neurological damage caused by intracerebral hemorrhage transformation.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the application of histidine-rich glycoprotein in predicting, preventing or treating hemorrhagic transformation after thrombolytic therapy in patients with cerebral infarction. Background Art

[0002] Recombinant tissue plasminogen activator (rtPA, commercially known as alteplase) is the only intravenous thrombolytic drug approved to date for the treatment of acute ischemic stroke (AIS, also known as acute cerebral infarction). However, rtPA has a strict time window requirement and is limited to use within 4.5 hours of onset. It also has side effects, especially the most serious one, which is hemorrhagic transformation (HT) after medication. It can cause worsening brain damage in patients and even death in severe cases. It is common in patients who receive thrombolysis a long time after onset. Therefore, only a small number of patients who arrive at the hospital in time have the opportunity to receive thrombolytic therapy. Moreover, since it takes 30-40 minutes for patients to undergo preliminary examinations (such as blood tests and head CT scans) after arriving at the hospital, doctors and patients are concerned about the risk of thrombolysis for patients who are admitted to the hospital close to the time window (3.5-4.5 hours after onset), which greatly reduces the thrombolytic rate. The actual benefits of delayed thrombolysis are reduced in patients, and the risk of hemorrhagic transformation is greatly increased. Therefore, it is of great clinical significance to explore markers for estimating bleeding risk and to use medication in a timely manner so that patients can prevent or reduce bleeding transformation after thrombolysis, increase the benefits of delayed thrombolysis patients, and reduce the risk of thrombolysis.

[0003] The causes of hemorrhagic transformation after thrombolysis include decreased integrity of the blood-brain barrier, dissolution of the extravascular matrix, and infiltration of immune cells, mainly neutrophils, into the brain. Among them, neutrophil infiltration aggravates the release of inflammatory factors and injury-related factors, further destroys the blood-brain barrier, and aggravates nerve damage. Due to the occurrence and development of cerebral infarction and the changes in the secondary coagulation / fibrinolysis system caused by thrombolysis, it plays an important regulatory role in cerebral infarction-post-infarction cerebral hemorrhage. Therefore, there is an urgent need for a drug based on endogenous regulatory proteins as an immune and coagulation / fibrinolysis regulator to maintain immune and coagulation homeostasis, so that rtPA can exert its anticoagulant effect while reducing the risk of bleeding. Summary of the invention

[0004] Problem that the invention aims to solve

[0005] One of the side effects of rtPA, a drug for treating cerebral infarction, is that it causes intracerebral hemorrhagic transformation in patients after taking the drug, which can lead to death in severe cases. However, there is currently a lack of effective markers or reliable evaluation methods to determine whether AIS patients will experience hemorrhagic transformation after receiving rtPA thrombolysis, and there is currently a lack of effective prevention and treatment methods for hemorrhagic transformation. In this regard, the present invention has found through research that histidine-rich glycoprotein can be used as a marker to effectively predict outcomes related to hemorrhagic transformation after thrombolytic treatment of cerebral infarction patients, and it can also be used to reduce hemorrhagic transformation after thrombolytic treatment of cerebral infarction, and assist in extending the time range of rtPA administration, so the present invention provides the use of histidine-rich glycoprotein in predicting, preventing or treating hemorrhagic transformation after thrombolytic treatment of cerebral infarction patients.

[0006] Solutions for solving problems

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] , Use of histidine-rich glycoprotein in any of the following (a1) to (a3):

[0009] (a1) preparing a kit for predicting the risk of hemorrhagic transformation in subjects with cerebral infarction after thrombolytic therapy;

[0010] (a2) preparing a medicament for preventing hemorrhagic transformation after thrombolytic therapy in subjects with cerebral infarction;

[0011] (a3) preparing a medicament for treating hemorrhagic transformation after thrombolytic therapy in subjects with cerebral infarction;

[0012] Preferably, the histidine-rich glycoprotein comprises a sequence as shown in any one of the following (b1) to (b3):

[0013] (b1) the amino acid sequence shown in SEQ ID NO.1;

[0014] (b2) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence as shown in SEQ ID NO.1 and has histidine-rich glycoprotein activity;

[0015] (b3) a sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 1 and having histidine-rich glycoprotein activity.

[0016] , The use according to [1], characterized in that the thrombolytic therapy is a treatment of administering recombinant tissue plasminogen activator to the subject with cerebral infarction;

[0017] Preferably, the recombinant tissue plasminogen activator comprises a sequence as shown in any one of the following (c1) to (c3):

[0018] (c1) the amino acid sequence shown in SEQ ID NO.2;

[0019] (c2) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO.2 and has tissue-type plasminogen activator activity;

[0020] (c3) a sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 2 and having tissue-type plasminogen activator activity.

[0021] , The use according to [1] or [2], characterized in that the thrombolytic therapy causes at least one of the following symptoms: neutrophil NETosis, proinflammatory cytokine release, ROS production, blood-brain barrier damage, cerebral hemorrhage and neutrophil infiltration of the central nervous system.

[0022] , The use according to any one of [1] to [3], characterized in that the subject comprises a mammal;

[0023] Preferably, the mammal comprises a human.

[0024] , the use according to any one of [1] to [4], characterized in that the kit is used to detect the content of histidine-rich glycoprotein in the sample derived from the subject with cerebral infarction;

[0025] Preferably, the kit comprises a reagent for detecting the content of histidine-rich glycoprotein in the sample;

[0026] More preferably, the agent comprises an antibody.

[0027] , The use according to [5], characterized in that the sample comprises a first sample and a second sample, the first sample is a sample taken 0.5 to 1.5 hours before thrombolytic therapy for the subject with cerebral infarction, and the second sample is a sample taken 0.5 to 2.5 hours after thrombolytic therapy for the subject with cerebral infarction;

[0028] Preferably, if the content of histidine-rich glycoprotein in the second sample is not increased compared to the first sample, it indicates that the subject with cerebral infarction receiving thrombolytic therapy corresponding to the sample has a risk of hemorrhagic transformation.

[0029] , The use according to [5] or [6], characterized in that the sample comprises a blood sample;

[0030] Preferably, the blood sample comprises plasma, serum or whole blood.

[0031] , Use of a drug combination in any one of the following (d1) to (d3):

[0032] (d1) preparing a medicament for treating cerebral infarction;

[0033] (d2) preparing a drug for treating cerebral infarction complicated with hemorrhagic transformation;

[0034] (d3) preparing a drug for treating cerebral infarction and preventing hemorrhagic transformation;

[0035] Wherein, the drug combination comprises a thrombolytic drug and a histidine-rich glycoprotein.

[0036] , The use according to [8], characterized in that the histidine-rich glycoprotein comprises a sequence as shown in any one of the following (b1) to (b3):

[0037] (b1) the amino acid sequence shown in SEQ ID NO.1;

[0038] (b2) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence as shown in SEQ ID NO.1 and has histidine-rich glycoprotein activity;

[0039] (b3) a sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 1 and having histidine-rich glycoprotein activity.

[0040] , The use according to [8] or [9], characterized in that the thrombolytic drug comprises recombinant tissue plasminogen activator;

[0041] Preferably, the recombinant tissue plasminogen activator comprises a sequence as shown in any one of the following (c1) to (c3):

[0042] (c1) the amino acid sequence shown in SEQ ID NO.2;

[0043] (c2) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO.2 and has tissue-type plasminogen activator activity;

[0044] (c3) a sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 2 and having tissue-type plasminogen activator activity.

[0045] Effects of the Invention

[0046] The results of the present invention show that after thrombolysis of cerebral infarction subjects with thrombolytic drugs such as rtPA, the level of histidine-rich glycoprotein in individuals without hemorrhagic transformation increases, while in individuals with hemorrhagic transformation, histidine-rich glycoprotein does not increase. Histidine-rich glycoprotein can be used as a marker to predict the outcomes related to hemorrhagic transformation in patients after thrombolytic treatment of cerebral infarction. Further, serological review is performed on patients with cerebral infarction within a period of time after cerebral infarction or as soon as possible (for example, 0.5 to 1.5 hours) after the end of thrombolysis, and the expression of histidine-rich glycoprotein is detected. It is of great clinical significance to take drug prevention as soon as possible, reduce the probability of hemorrhagic transformation in thrombolytic treatment of patients with cerebral infarction, and improve the actual benefits of patients with thrombolytic treatment. At the same time, the present invention also found that histidine-rich glycoprotein can inhibit rtPA-induced neutrophil NETosis and NETs formation, proinflammatory cytokine release, ROS production, blood-brain barrier damage, and neutrophil infiltration of the central nervous system, which can effectively reduce the intracerebral hemorrhagic transformation caused by delayed administration of rtPA and the neurological damage caused by intracerebral hemorrhagic transformation.

[0047] On this basis, the present invention provides the use of histidine-rich glycoprotein in the preparation of a kit for predicting the risk of hemorrhagic transformation after thrombolytic therapy in subjects with cerebral infarction, and the use of histidine-rich glycoprotein in the preparation of a drug for preventing and / or treating hemorrhagic transformation after thrombolytic therapy in subjects with cerebral infarction. By detecting the changes in the content of histidine-rich glycoprotein in blood samples of subjects with cerebral infarction, it is possible to accurately predict whether patients in the acute phase of cerebral infarction treatment have a risk of hemorrhagic transformation, and to provide a more comprehensive safety guarantee for thrombolytic therapy in patients with cerebral infarction. At the same time, histidine-rich glycoprotein can be used as a drug in combination with thrombolytic drugs such as rtPA, which can effectively reduce the risk of intracerebral hemorrhagic transformation while treating cerebral infarction, and assist in extending the time range of rtPA administration, which is of great significance to the clinical treatment of cerebral infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1A These are the results of differentially expressed proteins detected in the patients before and after thrombolysis in Example 1.

[0049] Figure 1B This is the KEGG classification analysis result of the differentially expressed proteins before and after thrombolysis in the patients in Example 1.

[0050] Figure 1C This is the KEGG pathway analysis result of the differentially expressed proteins before and after thrombolysis in the patients in Example 1.

[0051] Figure 1D This is the result of GO enrichment analysis of differentially expressed proteins in patients before and after thrombolysis in Example 1.

[0052] Figure 1EThis is the logarithmic transformation result of the Q value of the differentially expressed proteins before and after thrombolysis in Example 1.

[0053] Figure 1F This is the ranking result of the fold change of differentially expressed proteins before and after thrombolysis in Example 1.

[0054] Figure 2A This is the PPI network of differentially expressed proteins in patients before and after thrombolysis in Example 1.

[0055] Figure 2B These are the top 20 central proteins in the PPI network of differentially expressed proteins in patients before and after thrombolysis in Example 1.

[0056] Figure 2C This is the cluster network of differentially expressed proteins in patients before and after thrombolysis in Example 1.

[0057] Figure 3A These are the test results of HRG levels in different patients in Example 2.

[0058] Figure 3B These are the test results of elevated HRG levels in different patients (combined HT and non-HT) in Example 2.

[0059] Figure 3C The elevated HRG levels in different patients in Example 2 are used to predict the bleeding risk of patients after rtPA treatment.

[0060] Figure 4A : Calcein AM staining results of neutrophils in culture medium containing different concentrations of rtPA in Example 3.

[0061] Figure 4B : Statistical results of neutrophil mortality in culture medium containing different concentrations of rtPA in Example 3.

[0062] Figure 4C : Schematic diagram of HRG concentration in Example 3.

[0063] Figure 4D : Calcein AM staining results of neutrophils in culture medium containing different concentrations of HRG in Example 3.

[0064] Figure 4E : Statistical results of neutrophil mortality in culture medium containing different concentrations of HRG in Example 3.

[0065] Figure 5A : The detection results of NETosis in different groups of neutrophils in Example 4; wherein the arrow indicates NETosis.

[0066] Figure 5B: Statistical results of the H3Cit ratios of neutrophils in different groups in Example 4.

[0067] Figure 5C : Statistical results of neutrophil MPO ratios in different groups in Example 4.

[0068] Figure 5D : Statistical results of NET formation ratios of neutrophils in different groups in Example 4.

[0069] Figure 5E : The detection results of TNF-α content in different groups in Example 4.

[0070] Fig. 5F : The detection results of IL-8 content in different groups in Example 4.

[0071] Figure 5G : The detection results of ROS content in different groups in Example 4.

[0072] Fig. 6A : The staining results of the neutrophil cytoskeleton protein F-actin in different groups in Example 5.

[0073] Figure 6B : Statistical results of the aggregation of neutrophil cytoskeleton protein F-actin in different groups in Example 5.

[0074] Figure 6C : Schematic diagram of Transwell experiment in Example 5.

[0075] Fig.6D : Statistical results of the mobility of neutrophils in different groups at 2 h in Example 5.

[0076] Fig. 6E : Statistical results of the mobility of neutrophils in different groups at 4 h in Example 5.

[0077] Fig. 6F : Statistical results of the mobility of neutrophils in different groups at 8 h in Example 5.

[0078] Fig. 7A : Experimental schematic diagram in Example 6.

[0079] Figure 7B : Bederson scoring results of different groups of mice in Example 6.

[0080] Figure 7C : Statistical results of removal time of different groups of mice in Example 6.

[0081] Fig.7D : The right turn percentage of mice in different groups in Example 6.

[0082] Fig. 7E : The observation results of intracranial hemorrhage in different groups of mice in Example 6.

[0083] Figure 7F : Statistical results of intracranial hemoglobin content in different groups of mice in Example 6.

[0084] Figure 7G : The immunofluorescence staining results of brain tissues of mice in different groups in Example 6.

[0085] Figure 7H : Statistical results of Ly6G ratios in brain tissues of different groups of mice in Example 6. DETAILED DESCRIPTION

[0086] The following is a detailed description of the present invention. The technical features described below are described based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples.

[0087] I. Definition of Terms

[0088] In this specification, “a” or “an” may refer to “one”, or may refer to “one or more”, “at least one”, or “one or more than one”.

[0089] In this specification, "include", "have", "include" or "contain" and any variation thereof are intended to cover non-exclusive inclusions. At the same time, "include", "have", "include" or "contain" and any variation thereof may also represent closed, exclusive inclusions.

[0090] In this specification, "substantially" means that the numerical range and parameters of the present invention are approximate values, and the present invention has presented specific relevant values ​​as accurately as possible. Unless otherwise clearly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are approximate values, which usually refer to the actual value within ±5%, ±3%, ±1% or ±0.5% of a specific value or range.

[0091] In the present specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes cases where the event occurs and cases where it does not occur.

[0092] In this specification, a "patient" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates such as monkeys), rabbits, and rodents (such as mice and rats).

[0093] In this specification, "cerebral infarction", "stroke", "acute cerebral infarction" and "acute ischemic stroke" have the same meaning, and all refer to brain tissue necrosis caused by stenosis or occlusion of the brain's blood supply arteries (carotid artery and vertebral artery) and insufficient blood supply to the brain.

[0094] In this specification, "AUC" is an abbreviation for "area under the curve". Specifically, it refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a graph of the true positive rate relative to the false positive rate for different possible cut points of a diagnostic test. It shows the trade-off between sensitivity and specificity according to the selected cut point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test.

[0095] In this specification, "neutrophil reticular extracellular trap formation (NETosis)" refers to a process in which neutrophils release extracellular reticular DNA and various proteins.

[0096] In this specification, "treatment" means: after suffering from a disease (e.g., hemorrhagic transformation after thrombolytic therapy of cerebral infarction), the subject is exposed to (e.g., administered) the histidine-rich glycoprotein of the present invention, thereby reducing the symptoms of the disease (e.g., hematoma volume, neurological damage, etc.) compared to when not exposed, and does not mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means: the body has symptoms of the disease.

[0097] In this specification, "prevention" means: before suffering from a disease (such as hemorrhagic transformation after thrombolytic treatment of cerebral infarction), by exposing the subject to (such as administering) the histidine-rich glycoprotein described in the present invention, the symptoms after suffering from the disease (such as hematoma volume, neurological damage, etc.) are reduced compared with when not in contact, and it does not mean that the disease must be completely suppressed.

[0098] As used herein, "administering," "administering," and "treating," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, experimental subject, cell, tissue, organ, or biological fluid.

[0099] In this specification, "effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease (e.g., hemorrhagic transformation after thrombolytic therapy of cerebral infarction) such as hematoma volume, neurological damage, etc.). An effective amount also means an amount sufficient to allow or promote diagnosis. The effective amount for a specific patient or veterinary subject may vary according to factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0100] In this specification, the terms "sequence identity" and "percentage identity" refer to the percentage of identical nucleotides or amino acids between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides, inserted nucleotides, or missing nucleotides. The polypeptides may differ at one position, for example, by containing different amino acids, inserted amino acids, or missing amino acids. Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0101] In the present specification, amino acid deletion may refer to deleting one or more amino acids from an amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0102] In this specification, amino acid addition may refer to adding one or more amino acids at the C-terminus, N-terminus, or any position between the C-terminus and the N-terminus of an amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0103] In the present specification, amino acid substitution may refer to the replacement of an amino acid at a certain position of an amino acid sequence by another amino acid, as long as the altered sequence retains the activity of the original amino acid sequence in whole or in part. Amino acid substitution may be a conservative amino acid substitution, thereby producing a conservative variant peptide, that is, compared with the original amino acid sequence, several amino acids are replaced by amino acids with similar or similar properties to form a peptide. Exemplarily, these conservative variant peptides can be produced according to the following amino acid substitutions: substitution of Ala by Val, Leu or Ile, substitution of Arg by Lys, Gln, Asn or His, substitution of Asn by Gln, His, Lys or Arg, substitution of Asn by Glu or Asn, substitution of Cys by Ser or Ala, substitution of Gln by Asn or Glu, substitution of Glu by Asp or Gln, substitution of Gly by Ala, substitution of His by Asn, Lys, Gln or Arg, substitution of Leu, Met, Ala, Val, Phe or norleucine by The amino acid substitutions may also be non-conservative amino acid substitutions.

[0104] Unless otherwise defined, other 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.

[0105] II. The present invention is mainly obtained through the following research

[0106] The researchers of the present invention gave thrombolytic therapy (rtPA administration therapy) to patients with acute ischemic cerebral infarction in the acute phase, and unexpectedly found that the level of histidine-rich glycoprotein increased 1 hour after the end of the treatment; while in patients with hemorrhagic transformation after thrombolysis, the increase of histidine-rich glycoprotein was not significant. Further, the present invention realized that histidine-rich glycoprotein may be used to predict the risk of hemorrhagic transformation in the later stage of thrombolytic therapy in patients with cerebral infarction, and the increase of histidine-rich glycoprotein may help to reduce the risk of hemorrhagic transformation in the later stage. Further, the researchers of the present invention constructed an animal model of thrombolytic therapy for acute cerebral infarction combined with hemorrhagic transformation. After administering histidine-rich glycoprotein, it was found that the hemorrhagic transformation was alleviated and the neurological function score was improved. Further, the present invention found through research and analysis that histidine-rich glycoprotein can inhibit the symptoms of neutrophil NETosis, neutrophil inflammatory factor release, and neutrophil crossing the blood-brain barrier caused by rtPA, and reduce hemorrhagic transformation and the neural damage caused by it.

[0107] III. Uses of Histidine-Rich Glycoproteins

[0108] The present invention provides the use of histidine-rich glycoprotein in any one of the following (a1) to (a3):

[0109] (a1) preparing a kit for predicting the risk of hemorrhagic transformation in subjects with cerebral infarction after thrombolytic therapy;

[0110] (a2) preparing a medicament for preventing hemorrhagic transformation after thrombolytic therapy in subjects with cerebral infarction;

[0111] (a3) preparing a drug for treating hemorrhagic transformation after thrombolytic therapy in a subject with cerebral infarction.

[0112] The present invention found that histidine-rich glycoprotein can be used as a marker to predict the outcomes related to hemorrhagic transformation in patients with cerebral infarction after thrombolytic therapy. At the same time, histidine-rich glycoprotein prevents and / or treats hemorrhagic transformation in subjects with cerebral infarction after thrombolytic therapy.

[0113] In some embodiments, the histidine-rich glycoprotein comprises a sequence as shown in any one of the following (b1) to (b3):

[0114] (b1) the amino acid sequence shown in SEQ ID NO.1;

[0115] (b2) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence as shown in SEQ ID NO.1 and has histidine-rich glycoprotein activity;

[0116] (b3) a sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 1 and having histidine-rich glycoprotein activity.

[0117] In some preferred embodiments, the histidine-rich glycoprotein comprises the amino acid sequence shown in SEQ ID NO.1.

[0118] In some more preferred embodiments, the amino acid sequence of the histidine-rich glycoprotein is as shown in SEQ ID NO.1.

[0119] In some embodiments, the thrombolytic therapy is a therapy in which recombinant tissue plasminogen activator is administered to the subject with cerebral infarction.

[0120] In some specific embodiments, the recombinant tissue plasminogen activator comprises a sequence as shown in any one of the following (c1) to (c3):

[0121] (c1) the amino acid sequence shown in SEQ ID NO.2;

[0122] (c2) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO.2 and has tissue-type plasminogen activator activity;

[0123] (c3) a sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 2 and having tissue-type plasminogen activator activity.

[0124] In some preferred embodiments, the recombinant tissue plasminogen activator comprises the amino acid sequence shown in SEQ ID NO.2.

[0125] In some more preferred embodiments, the amino acid sequence of the recombinant tissue plasminogen activator is shown as SEQ ID NO.2.

[0126] In some embodiments, the thrombolytic therapy is delayed thrombolytic therapy, that is, thrombolytic therapy performed after a period of time after cerebral infarction occurs.

[0127] In some specific embodiments, the thrombolytic therapy is thrombolytic therapy performed greater than or equal to 3 hours after the onset of cerebral infarction.

[0128] In some specific embodiments, the thrombolytic therapy is thrombolytic therapy that occurs within 4.5 hours of cerebral infarction.

[0129] In some embodiments, the thrombolytic therapy causes at least one of the following symptoms: neutrophil reticular extracellular trap generation (NETosis), proinflammatory cytokine release, reactive oxygen species (ROS) generation, blood-brain barrier damage, cerebral hemorrhage, and neutrophil infiltration of the central nervous system.

[0130] In some specific embodiments, the hemorrhagic transformation of the subject with cerebral infarction after thrombolytic therapy includes neurological damage caused by intracerebral hemorrhage.

[0131] The present invention finds that histidine-rich glycoprotein can inhibit rtPA-induced neutrophil NETosis and neutrophil extracellular traps (NETs) formation, pro-inflammatory cytokine release (including but not limited to IL-8 and TNF-α), ROS production, blood-brain barrier damage, cerebral hemorrhage and neutrophil infiltration of the central nervous system, and can effectively reduce rtPA-induced intracerebral hemorrhage transformation and neurological function damage caused by delayed administration of rtPA.

[0132] In some embodiments, the subject comprises a mammal; preferably, the mammal comprises a human.

[0133] In some preferred embodiments, the subject is a human.

[0134] In some embodiments, the kit is used to detect the content of histidine-rich glycoprotein in a sample derived from the subject with cerebral infarction.

[0135] In some embodiments, the kit comprises reagents for detecting the content of histidine-rich glycoprotein in the sample; illustratively, the reagents include but are not limited to antibodies.

[0136] In some specific embodiments, the kit detects the content of histidine-rich glycoprotein in the sample using the principle of enzyme-linked immunosorbent assay.

[0137] In some embodiments, the sample includes a first sample and a second sample; the first sample is a sample taken 0.5 to 1.5 hours before thrombolytic therapy for the subject with cerebral infarction, preferably 1 hour; the second sample is a sample taken 0.5 to 2.5 hours after thrombolytic therapy for the subject with cerebral infarction, preferably 0.5 to 1.5 hours, and more preferably 1 hour.

[0138] Predictive assessment of bleeding risk as soon as possible after patients with cerebral infarction receive thrombolytic therapy can effectively improve the actual benefits of thrombolytic therapy for patients with cerebral infarction.

[0139] In some embodiments, if the content of histidine-rich glycoprotein in the second sample is not increased compared to the first sample, it indicates that the subject receiving thrombolytic therapy for cerebral infarction corresponding to the sample has a risk of hemorrhagic transformation.

[0140] In some embodiments, the sample comprises a blood sample.

[0141] In some embodiments, the blood sample comprises plasma, serum, or whole blood;

[0142] In some preferred embodiments, the blood sample is plasma.

[0143] The present invention also provides a use of a drug combination in any one of the following (d1) to (d3):

[0144] (d1) preparing a medicament for treating cerebral infarction;

[0145] (d2) preparing a drug for treating cerebral infarction complicated with hemorrhagic transformation;

[0146] (d3) preparing a drug for treating cerebral infarction and preventing hemorrhagic transformation;

[0147] Wherein, the drug combination comprises a thrombolytic drug and a histidine-rich glycoprotein.

[0148] Histidine-rich glycoprotein can be used as a drug in combination with thrombolytic drugs such as rtPA. It can effectively reduce the risk of intracerebral hemorrhage while treating cerebral infarction and help extend the time range of rtPA administration, which is of great significance for the clinical treatment of cerebral infarction.

[0149] IV. Amino Acid Sequences in the Present Invention

[0150] Amino acid sequence of histidine-rich glycoprotein (SEQ ID NO.1):

[0151] MKALIAALLLITLQYSCAVSPTDCSAVEPEAEKALDLINKRRRDGYLFQLLRIADAHLDRVENTTVYYLVLDVQESDCSVLSRKYWNDCEPPDSRRPSEIVIGQCKVIATRHSHESQDLRVIDFNCTTSSVSSALANTKDSPVLIDFFEDTERYRKQANKALEKYKEENDDFASFRVDRIERVARVRGGEGTGYFVDFSVRNCPRHHFPRHPNVFGFCRADLFYDVEALDLESPKNLVINCEVFDPQEHENINGVPPHLGHPFHWGGHERSSTTKPPFKPHGSRDHHHPHKPHEHGPPPPPDERDHSHGPPLPQGPPPLLPMSCSSCQHATFGTNGAQRHSHNNNSSDLHPHKHHSHEQHPHGHHPHAHHPHEHDTHRQHPHGHHPHGHHPHGHHPHGHHPHGHHPHCHDFQDYGPCDPPPHNQGHCCHGHGPPPGHLRRRGPGKGPRPFHCRQIGSVYRLPPLRKGEVLPLPEANFPSFPLPHHKHPLKPDNQPFPQSVSESCPGKFKSGFPQVSMFFTHTFPK。

[0152] Amino acid sequence of recombinant tissue-type plasminogen activator (SEQ ID NO.2):

[0153] .

[0154] Example

[0155] The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out under normal conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are all commercially available conventional products.

[0156] Example 1

[0157] Experimental methods:

[0158] (1) Patient cohort construction and plasma sample collection

[0159] In the discovery cohort, a total of 10 AIS patients who received rtPA treatment were recruited from June 2017 to July 2019. Blood samples were drawn before the start of emergency rtPA treatment and within 1 hour after the completion of treatment (recorded as before rtPA and after rtPA, respectively), and plasma samples were separated for proteomic analysis.

[0160] (2) Plasma protein extraction, enrichment and digestion

[0161] Ethyl ketone was added to the plasma sample, and the mixture was centrifuged to separate the supernatant. The resulting precipitate was air dried, and then a lysis buffer (200 μl) containing urea (420 g / L), thiourea (152 g / L), SDS (2 g / L) and Tris (2.4 g / L) was added to redissolve the precipitate. Subsequently, dithiothioethanol and iodoacetate were added at room temperature under dark conditions for alkylation reaction, and the protein concentration in the supernatant was determined using a Bradford kit (Bio-Rad, Hercules, CA, USA). The diluted plasma protein mixture was loaded and passed through an SPE C18 column at a rate of 1 drop per second. Subsequently, non-specifically bound proteins were eluted and enriched proteins were eluted. Each sample was diluted with an appropriate amount of protein solution (50 mM ammonium bicarbonate) and mixed with an appropriate amount of sample buffer (Tris HCl (pH 6.8, 1.0 M) 2 ml, Milli-Q H2O 3 ml, SDS 0.8 g, DTT 0.568 g, completely dissolved, glycerol 4 ml mixed, then bromophenol blue (10%) 50 μl, Milli-Q H2O to 10 ml) for electrophoresis. Subsequently, trypsin was added to the protein solution at a ratio of 1:20 (w / w) and incubated at 37°C for 14-16 hours.

[0162] (3) High pH reverse phase separation

[0163] Peptides from each sample were mixed equally and diluted with 2 ml of mobile phase A (5% acetonitrile, pH 9.8) before injection. A Shimadzu LC-20AB HPLC system was used, equipped with a Gemini high pH C18 column (5 μm, 4.6×250 mm). The sample was separated by column and eluted at a flow rate of 1 ml / min using the following gradient: 5% mobile phase B (95% acetonitrile, pH 9.8) in 10 min, 5%-35% mobile phase B in 40 min, 35%-95% mobile phase B in 1 min, mobile phase B in 3 min, and finally equilibrated to 5% mobile phase B in 10 min. The eluent was collected every 1 min. Finally, all eluents were combined into 10 fractions, frozen and dried.

[0164] (4) Data-dependent acquisition (DDA) and data-independent acquisition (DIA) analysis by nano-liquid chromatography tandem mass spectrometry (LC-MS / MS)

[0165] The dried peptide samples were redissolved in mobile phase A (2% acetonitrile, 0.1% FA) and centrifuged, and the supernatant was collected for injection. The sample separation was performed using a Thermo UltiMate 3000 UHPLC liquid chromatograph. First, the sample was enriched and desalted in a trap column, and then entered a tandem self-packed C18 column for separation. Subsequently, it was separated by an effective gradient, ionized by nanoESI, and injected into a Q-Exactive HF X tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) for DDA and DIA analysis. The mass spectrometry parameters were set as follows: for DDA mode: ion source voltage 1.9 kV; MS scan range 350-1,500 m / z; MS resolution 120,000, maximum injection time (MIT) 50 ms; MS / MS collision type HCD, collision energy NCE 28; MS / MS resolution 30,000, MIT 100 ms, dynamic exclusion duration 30 s. The starting m / z value of the MS / MS scan was fixed at 100. The precursor ions for the MS / MS scan met the following conditions: charge range 2+ to 6+, and the first 20 precursor ions with intensities exceeding 2E4. The AGC used was 3E6 for MS and 1E5 for MS / MS. For the DIA analysis, the same nano-LC system and gradient as for the DDA analysis were used. The main settings were as follows: ion source voltage 1.9–2 kV; MS scan range 400–1,250 m / z; MS resolution 120,000, MIT 50 ms; MS / MS scans from 400 to 1,250 m / z, equally divided into 45 consecutive windows. The MS / MS collision type HCD MIT was in automatic mode. The fragment ions were scanned in the Orbitrap with an MS / MS resolution of 30,000, collision energies in distribution mode: 22.5, 25, and 27.5, and AGC 1E6.

[0166] (5) Protein identification, quantitative analysis and bioinformatics analysis

[0167] The DDA data were used as a spectral library for subsequent DIA analysis for protein identification using MaxQuant software (version 1.5.3.30). The identified peptides that met FDR ≤ 1% were used to construct the final spectral library. The parameters were set as follows: enzyme: trypsin; minimum peptide length: 7; PSM level FDR and protein FDR: 0.01; fixed modification: carboxamidomethyl (C); variable modification: oxidation (M); acetyl (protein N-terminus); glutamine->malonamide amide (N-terminal Q); deamidation (NQ). The DIA data were retained time aligned using iRT peptides. Then, based on the target pseudo-target model suitable for SWATH-MS, a false positive control with an FDR of 1% was performed to obtain significant quantitative results. Finally, the MSstats software package was used to evaluate statistically significant differences in the proteome. Differential protein screening was performed according to the criteria of fold change>2 and p value<0.05.

[0168] The online DAVID software was used to perform GO (Gene Ontology) analysis on the annotation functions of proteins. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database was used to perform pathway analysis on the identified plasma proteins. The STRING database (http: / / string-db.org) was used for protein–protein interaction (PPI) network analysis.

[0169] Experimental results:

[0170] Based on the protein quantification results, differentially expressed proteins (DEPs) before and after thrombolysis were determined (e.g. Figure 1A According to fold change>1.5 and Q value<0.05, a total of 119 DEPs were identified, including 84 up-regulated proteins and 35 down-regulated proteins. KEGG classification analysis of DEPs revealed that they were mainly involved in the immune system (such as Figure 1B KEGG pathway analysis further showed that DEPs were significantly enriched in pathogenic infection, cell-cell junction, platelet activation, complement and coagulation cascades, and TGF-β signaling pathways (as shown in Figure 2A). Figure 1C GO enrichment analysis showed that proteins involved in certain biological processes were significantly enriched in DEPs, including mitotic cell cycle, microtubule-related processes, negative regulation of endothelial cell chemotaxis, platelet activation, and fibrinolytic processes (e.g. Figure 1D Based on the logarithmic transformation results of -Q values, the top 20 most significantly upregulated proteins were identified, among which HRG was the most significantly increased (as shown in Figure 1EAs shown in the figure, HRG is one of the proteins with significant increase in fold change ranking (as shown in the figure). Figure 1F shown).

[0171] Figure 2A The PPI network of plasma DEPs between individuals before and after rtPA treatment is shown. The p-value of DEPs is represented by the node size, and the thickness of the edge indicates the degree of interaction between DEPs. The color of the node distinguishes upregulated (red) and downregulated (blue) DEPs. Protein interaction (PPI) network analysis was performed on the differentially expressed proteins (DEPs) identified above using the STRING database. After removing proteins with no recognized symbolic names, the PPI network involved 49 nodes and 196 edges. The top 20 hub proteins (hub DEPs) identified using the CytoHubba plugin included ACTB, FN1, CFL1, ACTA2, RHOA, TAGLN, YWHAZ, THBS1, HSP90AB1, TUBA1C, TUBA1B, TUBB, KNG1, ITIH4, TAGLN, MYL6, FGA, FGG, HRG, TPM4, and F5 (e.g. Figure 2B shown). Figure 2C The cluster network identified by the MCODE plugin is shown, one of which is mainly related to fibrinolysis and inflammatory response. Seven of the top 20 hub genes were included in this cluster, namely HRG, FGG, FGA, F5, KNG1, THBS1, and ITIH4. HRG served as a central node in the interaction network connecting proteins related to fibrinolysis and inflammatory processes. Therefore, the increase in HRG after rtPA treatment in patients with acute cerebral infarction may have a role in regulating fibrinolysis and inflammatory response.

[0172] In summary, in the discovery cohort, 119 differentially expressed proteins were identified in the plasma of patients before and after thrombolysis using high-throughput protein profiling, among which HRG was the most significantly increased in the paired comparison before and after thrombolysis. Protein interaction (PPI) network analysis suggested that HRG was the central node connecting proteins related to fibrinolysis and inflammatory processes.

[0173] Example 2

[0174] Experimental methods:

[0175] (1) Patient cohort construction and plasma sample collection

[0176] In the validation cohort, a total of 78 AIS patients were recruited for ELISA testing from March 2020 to October 2023. 19 healthy volunteers served as healthy controls (recorded as HC). 61 AIS patients who received rtPA treatment (within 4.5h after AIS onset) were selected as the AIS+rtPA group (recorded as rtPA), while 17 patients who did not receive thrombolytic therapy or did not meet the conditions for thrombolytic therapy were selected as the AIS w / o rtPA group (recorded as stroke). In the AIS+rtPA group, plasma samples were collected before the start of rtPA infusion and 1 hour after the end (recorded as before and after, respectively). For patients in the AIS w / o rtPA group, blood samples were collected 3.5 hours and 6.5 hours after the onset of stroke symptoms.

[0177] (2) ELISA measurement of HRG using human plasma.

[0178] Plasma samples were collected from whole blood, and HRG levels were measured using a human HRG ELISA kit (ab277404, Abcam, Cambridge, UK). The absorbance was measured at 450 nm. First, the kit and samples were equilibrated at room temperature (25-28°C) for 120 min to fully equilibrate to room temperature. Standard wells, sample wells, and blank wells were set up. 100 μL of standard of different concentrations was added to each standard well; 100 μL of the sample to be tested was added to the sample well; 100 μL of sample diluent was added to the blank well. Incubate at room temperature for 2.5 hours with gentle shaking. After washing with washing solution, 100 μL of biotin-labeled detection antibody was added to each well, and incubated at room temperature for 60 min with gentle shaking. Discard the liquid, pat dry on absorbent paper, fill each well with 350 μL of washing solution, let it stand for 20 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times (you can also use a plate washer to wash the plate). Add 100 μL of the prepared streptavidin solution to each well. Incubate gently with shaking at room temperature for 45 minutes. After washing, add 100 μL of TMB substrate solution and incubate in the dark for 30 minutes. Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0179] Experimental results:

[0180] Figure 3A The results showed that the HRG level of patients with acute cerebral infarction who were given rtPA thrombolytic therapy increased after thrombolysis; while the HRG level of patients with acute cerebral infarction who were not given thrombolytic therapy did not increase, and there was no significant difference compared with the healthy control group. Figure 3BThe results showed that in the AIS+rtPA group, compared with the HRG level in the patient's plasma 1 hour before rtPA infusion, the HRG level of patients with hemorrhagic transformation (referred to as HT) did not increase 1 hour after rtPA infusion, while the HRG level of patients without hemorrhagic transformation (referred to as non-HT) increased 1 hour after rtPA infusion, and the change of HRG level before and after rtPA infusion between the HT group and the non-HT group was statistically significant (P<0.05). The above studies suggest that: 1 hour after rtPA thrombolysis, the HRG level in the patient's body increases, while the HRG level of individuals with hemorrhagic transformation does not increase. Figure 3C It was shown that in the AIS+rtPA group, the elevated HRG levels of patients in the hemorrhagic transformation group (n=7) and the non-hemorrhagic transformation group (n=54) were analyzed (i.e., the HRG level 1 hour after thrombolysis minus the HRG level 1 hour before thrombolysis). The ROC curve was drawn with 100%-specificity% as the horizontal axis and sensitivity% as the vertical axis. The AUC was 0.7460, indicating that the elevated HRG level in patients after rtPA treatment can be used to predict the bleeding risk of patients after rtPA treatment with good accuracy.

[0181] Example 3

[0182] Experimental methods:

[0183] (1) Isolation and culture of human peripheral blood neutrophils

[0184] Peripheral blood samples were collected from AIS patients within the thrombolytic time window. Neutrophils were extracted using a density gradient separation kit (P9040, Solebao Biotech, Beijing, China). The brief steps were as follows: Whole blood was centrifuged for 10 min and the plasma was discarded. Then, the separation reagent and blood cells were added to a 15-mL centrifuge tube in sequence and centrifuged at 800 g for 30 min. The neutrophil layer was retained according to the manufacturer's instructions and suspended with red blood cell lysis solution (R1010, Solebao Biotech, Beijing, China) for 15 min at 4°C. After centrifugation, the cells were resuspended in RPMI-1640 medium (PM150110, Pronocell Biotech, Wuhan, China) and cultured at 37°C. The purity of the isolated neutrophils was determined to be >98% by fluorescence-activated cell sorting analysis.

[0185] (2) Neutrophil survival rate detection

[0186] The neutrophil suspension was dispensed into a 96-well plate, with 100 μl per well (concentration of 5×10 5Cells / ml) were added with one of the following agents: PBS (denoted as control), rtPA (Boehringer Ingelheim) (2.5μg / ml, 5μg / ml, 10μg / ml, 20μg / ml, 40μg / ml or 100μg / ml), rtPA (20μg / ml) + HRG (Sino Biological) (0μM, 0.05μM, 0.10μM, 0.25μM, 0.50μM or 1μM) or lipopolysaccharides (LPS) (10μg / ml; Sigma-Aldrich). After stimulation and culture for 18 hours at 37°C and 5% CO2, neutrophils were stained with Hoechst 33342 (C0031, Solebio Biotech, Beijing, China) and calcein-AM (GC34061, Glpbio, California, USA), and then neutrophils were scanned. The calcein-negative neutrophils were counted using a fluorescence microscope (Biotek Cytation 5), and the neutrophil mortality rate was calculated according to the following formula: neutrophil mortality rate = calcein-negative cell count / total cell count.

[0187] Experimental results:

[0188] Figure 4A to Figure 4E The results showed that after adding rtPA to the neutrophil culture system, the neutrophil death rate increased with the increase of rtPA concentration; while in the neutrophil culture system containing 20μg / ml rtPA, the neutrophil death rate decreased when different concentrations of HRG were added.

[0189] Example 4

[0190] Experimental methods:

[0191] (1) In vitro NETosis assay

[0192] The isolated neutrophils were counted at 5×10 5Cells were seeded at a density of 10 cells / ml in 96-well plates and treated with PBS (control), rtPA (10 μg / ml), rtPA (10 μg / ml) + HRG (1 μM), or LPS (10 μg / ml) and incubated at 37°C for 2.5 hours. Then, neutrophils were fixed with 2% formaldehyde, blocked with 3% bovine serum albumin, and incubated with rabbit anti-histone H3 (H3Cit; 1:300; Abcam, MA, USA) and mouse anti-myeloperoxidase (MPO; 1:300; Invitrogen, USA), followed by incubation with Alexa-Fluor 488 goat anti-rabbit secondary antibody (1:1000; Invitrogen, CA, USA) and Alexa-Fluor 594 goat anti-mouse secondary antibody (1:1000; Invitrogen, CA, USA). Nuclear staining was performed using Hoechst 33342 (Solar Bio, Beijing, China). NETosis was observed by fluorescence microscopy (Biotek Cytation5, USA) and analyzed by ImageJ.

[0193] (2) ELISA measurement of IL-8, TNF-α and ROS was performed using the cell culture system supernatant.

[0194] The cells in the above culture system were centrifuged and the cell culture supernatant was collected. Human IL-8 (IL-8 / CXCL8) ELISA kit (MM-50812H1, Suzhou, China) and human TNF-α ELISA kit (MM-50812H1, Suzhou, China) were used to measure the IL-8 and TNF-α levels in the neutrophil culture supernatant respectively. Absorbance was measured at 450nm. According to the manufacturer's protocol, ROS levels were detected using ROS kit (KGT010, Nanjing, China Jinyu Biological).

[0195] Experimental results:

[0196] Figure 5A to Figure 5G It was shown that after adding rtPA to the culture system, neutrophil NETosis (H3cit, MPO co-staining) increased compared with the control group, and LPS was added as a positive control. Among them, after adding HRG to the rtPA-administered group, neutrophil NETosis decreased. In addition, the production of pro-inflammatory cytokines TNF-α, IL-8 and ROS in the culture system decreased.

[0197] Example 5

[0198] Experimental methods:

[0199] (1) Determination of total cellular F-actin

[0200] Freshly isolated neutrophils were distributed into 96-well plates (5 × 10 5 cells) and mixed with the following reagents: PBS (denoted as control), rtPA (10 μg / ml), rtPA (10 μg / ml) + HRG (0.5 μM), or LPS (10 μg / ml). After incubation at 37°C for 2.5 hours, neutrophils were fixed with 2% formaldehyde, treated with Triton X-100, blocked and then stained with Alexa-Fluor 488-conjugated mycotoxin (Thermo Fisher Scientific, Waltham, MA, USA) for 60 minutes. The level of F-actin was determined by fluorescence microscopy (Biotek Cytation 5, USA). All images used the same camera settings (exposure time, brightness, contrast and clarity) and set appropriate white balance according to the fluorescence filter. Images were acquired and analyzed using ImageJ. The average fluorescence density was determined from the fluorescence linear measurement of individual cells in randomly selected areas on each slide.

[0201] (2) In vitro blood-brain barrier (BBB) ​​model and permeability assay

[0202] Human brain microvascular endothelial cells (hCMECs; Zhongqiao Xinzhou Biotechnology, Shanghai, China) were cultured between the third and eighth passages using EGM-2MV medium (Lonza, Walkersville, MD, USA). Transwell system transmembrane inserts (pore size 3 mm, diameter 11 mm; Corning Life Science, Lowell, MA, USA) were coated with collagen IV (50 mg / ml) and fibronectin (30 mg / ml). hCMECs (density 2 × 10 5 Cells) were seeded on the upper side of the insert and cultured in complete medium containing 10% fetal bovine serum in EGM-2MV medium (Lonza, Walkersville, MD, USA) at 37°C for 3-4 days. When the cell monolayer reached complete sealing, the cell culture medium was replaced with glucose-free medium and exposed to hypoxia (95% nitrogen and 5% carbon dioxide) at 37°C for 4 hours. Then, neutrophils treated with PBS, rtPA or (rtPA+HRG (0.1μM, 0.5μM or 1μM) were added to the upper chamber after counting. The neutrophil counts in the lower chamber of each group were observed at 2 hours, 4 hours and 8 hours after incubation. The cell migration rate was calculated as follows: cell count in the lower chamber / initial number of cells added to the upper chamber.

[0203] Experimental results:

[0204] Figure 6A to Figure 6FThe results showed that F-actin staining was used to reflect the aggregation of neutrophil skeleton proteins in vitro, and it was found that the neutrophil skeleton protein F-actin aggregated after rtPA stimulation, suggesting that it was more likely to migrate. The transwell chamber coated with endothelial cells was further used to simulate the blood-brain barrier in vitro, and the migration experiment found that rtPA significantly induced neutrophil migration to the lower chamber, while the addition of HRG could inhibit neutrophil migration.

[0205] Example 6

[0206] Experimental methods:

[0207] (1) Photochemical embolization stroke model and rtPA thrombolytic therapy

[0208] Experimental animals were operated according to the standards of the Guide for the Care and Use of Laboratory Animals and in accordance with institutionally approved protocols. Male mice aged 8-10 weeks were used as a photochemical embolic stroke model. Mice were anesthetized by inhalation of 1%-1.5% isoflurane in a mixture of 30% oxygen and 70% nitrous acid. Body temperature was maintained at 37±0.5°C. The skin between the right eye and the right ear was cut, and the temporalis muscle was separated and retracted to expose the skull. The projection of the middle cerebral artery was found on the skull surface at the midpoint between the right eye and the right ear. The photosensitizing dye rose bengal (60 mg / kg; 330000, Sigma-Aldrich, St. Louis, MO, USA) was injected through the tail vein, and 5 minutes later, the projection area of ​​the middle cerebral artery was irradiated with a 3.5 mW green laser (540 nm) for 25 minutes. D-glucose (6 ml / kg, concentration of 50%, 158968, Sigma-Aldrich, St. Louis, MO, USA) was injected intraperitoneally 15 minutes before inducing stroke. At 1 hour and 5 hours after stroke, 1 mg / kg rtPA or saline (referred to as vehicle) was used as an intravenous push dose, and the latter was maintained by continuous intravenous infusion of 9 mg / kg for 30 minutes. At 5 hours after stroke, HRG (1 μM; 10836-H08H, SinoBiological, Beijing, China) or saline was injected intravenously.

[0209] (2) Neurological function assessment

[0210] Neurological deficits were measured using the Bederson score 24 hours after rtPA treatment. Rotarod and corner tests were used to assess sensorimotor function in each group.

[0211] (3) Quantitative measurement of cerebral hemorrhage

[0212] 24 hours after stroke, mice were transcardially perfused with PBS. Hemorrhage in the ischemic hemisphere brain tissue of each mouse was quantitatively measured using a hemoglobin content assay kit (BC5585, Beijing Solebao Biotechnology Co., Ltd.) according to the manufacturer's instructions.

[0213] (4) Neutrophil immunofluorescence staining

[0214] Mice were anesthetized and perfused with PBS, and brain tissue was collected. After fixation with 4% formaldehyde, the brain was cut into 8 μm thick sections by dehydration through 15% and 30% sucrose gradients. Blocking was performed with 3% bovine serum albumin and permeabilization with 0.3% Triton X-100. Mouse anti-Ly6G (1:200; Biolegend, CA, USA) was then added and incubated overnight at 4°C. Then, the sections were washed with PBS, incubated with Alexa-Fluor 488-conjugated secondary antibodies (1:1000; Invitrogen, CA, USA) at room temperature for 1.5 hours, washed again, and Monty's solution was added. Images were taken using a fluorescence microscope (Biotek Cytation 5, USA) and analyzed using ImageJ software.

[0215] Experimental results:

[0216] Figure 7A to Figure 7H The results showed that an animal model of acute cerebral infarction with middle cerebral artery occlusion was constructed using mice. rtPA was given 1 hour (early thrombolysis) and 5 hours (late thrombolysis) after thrombolysis. It was found that mice given rtPA at 5 hours had obvious intracranial hemorrhage and nerve damage. When HRG was given at the same time as rtPA at 5 hours, bleeding and nerve damage after cerebral infarction were significantly reduced. At the same time, immunofluorescence staining of brain tissue showed that the number of neutrophils infiltrating the brain increased significantly in the 5-hour thrombolysis group compared with the 1-hour thrombolysis and control groups, while it was significantly reduced in the 5-hour thrombolysis and HRG group.

Claims

1. Use of histidine-rich glycoprotein in any of the following (a1) to (a3): (a1) preparing a kit for predicting the risk of hemorrhagic transformation in subjects with cerebral infarction after thrombolytic therapy; (a2) preparing a medicament for preventing hemorrhagic transformation after thrombolytic therapy in subjects with cerebral infarction; (a3) preparing a drug for treating hemorrhagic transformation after thrombolytic therapy in a subject with cerebral infarction.

2. The use according to claim 1, characterized in that The histidine-rich glycoprotein comprises the amino acid sequence shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The thrombolytic therapy is a treatment in which recombinant tissue plasminogen activator is administered to the subject with cerebral infarction.

4. The use according to claim 3, characterized in that The recombinant tissue plasminogen activator comprises the amino acid sequence shown in SEQ ID NO.

2.

5. The use according to any one of claims 1 to 4, characterized in that: The thrombolytic therapy induces at least one of the following symptoms: neutrophil NETosis, pro-inflammatory cytokine release, ROS generation, blood-brain barrier damage, cerebral hemorrhage, and neutrophil infiltration of the central nervous system.

6. The use according to any one of claims 1 to 4, characterized in that The subject includes a mammal.

7. The use according to claim 6, characterized in that The mammals include humans.

8. The use according to any one of claims 1 to 4, characterized in that The kit is used for detecting the content of histidine-rich glycoprotein in the sample from the subject with cerebral infarction.

9. The use according to claim 8, characterized in that The kit comprises reagents for detecting the content of histidine-rich glycoprotein in the sample.

10. The use according to claim 9, characterized in that The reagents include antibodies.

11. The use according to claim 8, characterized in that The samples include a first sample and a second sample, wherein the first sample is a sample taken 0.5 to 1.5 hours before thrombolytic therapy for the subject with cerebral infarction, and the second sample is a sample taken 0.5 to 2.5 hours after thrombolytic therapy for the subject with cerebral infarction.

12. The use according to claim 11, characterized in that If the content of histidine-rich glycoprotein in the second sample is not increased compared to the first sample, it indicates that the subject with cerebral infarction receiving thrombolytic therapy corresponding to the sample has a risk of hemorrhagic transformation.

13. The use according to claim 8, characterized in that The sample comprises a blood sample.

14. The use according to claim 13, characterized in that The blood sample includes plasma, serum or whole blood.

15. Use of a drug combination in any one of the following (d1) to (d3): (d1) preparing a medicament for treating cerebral infarction; (d2) preparing a drug for treating cerebral infarction complicated with hemorrhagic transformation; (d3) preparing a drug for treating cerebral infarction and preventing hemorrhagic transformation; in, The drug combination comprises a thrombolytic drug and a histidine-rich glycoprotein.

16. The use according to claim 15, characterized in that The histidine-rich glycoprotein comprises the amino acid sequence shown in SEQ ID NO.

1.

17. The use according to claim 15 or 16, characterized in that The thrombolytic drug includes recombinant tissue plasminogen activator.

18. The use according to claim 17, characterized in that The recombinant tissue plasminogen activator comprises the amino acid sequence shown in SEQ ID NO.2.

Images