Application of MDL-1 in the preparation of brain protective drugs after ischemic stroke

Abstract

The present invention provides the use of MDL-1 in the preparation of brain protection drugs after ischemic stroke, belonging to the technical field of post-ischemic stroke treatment. The present invention proposes for the first time that MDL-1 has the effect of brain protection after ischemic stroke. In the present invention, MDL-1 regulates neuroinflammation after ischemic stroke by inhibiting the activation of microglia and astrocytes, converting neuroinflammation from pro-inflammatory to anti-inflammatory, and changing the inflammatory microenvironment in the brain after stroke. In addition, MDL-1 of the present invention can significantly reduce the apoptosis rate of cells on the side of cerebral infarction.

Description

Technical Field

[0001] The invention belongs to the technical field of post-ischemic stroke treatment, and in particular relates to the application of MDL-1 in the preparation of a brain protective drug for post-ischemic stroke. Background Art

[0002] Stroke is currently the main cause of increased disability and mortality in the middle-aged and elderly. It includes two types: hemorrhagic stroke and ischemic stroke, of which 85% are ischemic stroke patients. Patients with new cerebral infarction within 6 hours of onset can be treated with thrombolysis or thrombectomy to relieve blood flow blockage caused by thrombus or embolism, but less than 10% of patients meet the time window conditions. Patients with onset time beyond the time window can only be given brain protection treatment. Therefore, finding effective treatments to improve post-stroke pathological damage has been a research hotspot in stroke treatment in recent years. MDL-1 is a member of the C-type lectin domain family 5 A (CLEC5A), with a molecular weight of about 40 kDa, and is a glycosylated type II transmembrane protein. It consists of an extracellular region (ECD) of 165 amino acids (AA), a C-type lectin (CTL) domain and a membrane-paramembrane stem region, a 21-amino acid transmembrane region, and a 4-amino acid intracellular region. At present, there is no research related to the regulatory effect of MDL-1 on the inflammatory response after ischemic stroke. Summary of the invention

[0003] In view of this, the object of the present invention is to provide the use of MDL-1 in the preparation of brain protection drugs after ischemic stroke.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides application of MDL-1 in preparing a brain protection drug after ischemic stroke.

[0006] Preferably, the MDL-1 can significantly reduce the apoptosis rate of cells on the infarcted side.

[0007] Preferably, the MDL-1 can significantly inhibit the production of pro-inflammatory factors, including TNF-α, IL-1β and IL-6.

[0008] Preferably, the MDL-1 can significantly promote the production of anti-inflammatory factors, and the anti-inflammatory factors include IL-10 and Arg-1.

[0009] Preferably, the MDL-1 can significantly inhibit the production of inflammatory chemokines, including CCL-2, CCL-3, CCL-5 and CXCL-10.

[0010] Preferably, the MDL-1 regulates post-stroke neuroinflammation by inhibiting the activation of microglia and astrocytes.

[0011] The present invention also provides a brain protection drug after ischemic stroke, wherein the active ingredient of the drug includes MDL-1.

[0012] Preferably, the drug is an injectable preparation.

[0013] Preferably, the concentration of MDL-1 in the injection preparation is 0.25 μg / ml-0.5 μg / ml.

[0014] Preferably, the injection dosage of the injection preparation is 1 ml per kg body weight.

[0015] Beneficial effects of the present invention:

[0016] The present invention proposes for the first time that MDL-1 has a brain protective effect after ischemic stroke. In the present invention, MDL-1 regulates neuroinflammation after ischemic stroke by inhibiting the activation of microglia and astrocytes, converting neuroinflammation from pro-inflammatory to anti-inflammatory, and inhibiting leukocyte infiltration, thereby changing the inflammatory microenvironment in the brain after stroke. In addition, the MDL-1 of the present invention can significantly reduce the apoptosis rate of cells on the side of cerebral infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results show that MDL-1 plays a role in brain protection after ischemic stroke, where Figure A shows the behavioral experiment results one day after mouse modeling, Figure B shows the TTC staining results of cerebral infarction three days after mouse modeling, Figure C shows the cerebral infarction volume measured by ImageJ, Figure D shows the cell apoptosis results obtained by flow cytometry, and Figure E shows the statistical results of cell apoptosis;

[0018] Figure 2 The results show that MDL-1 has the effect of regulating neuroinflammation after ischemic stroke, wherein Figure A shows the results of pro-inflammatory factors in mice in the MDL-1 group, Figure B shows the results of anti-inflammatory factors in mice in the MDL-1 group, and Figure C shows the results of chemokines in mice in the MDL-1 group;

[0019] Figure 3 These are the results that MDL-1 has the function of regulating microglia and astrocytes, where Figure A shows the immunofluorescence results and cell counting results of microglia in the MDL-1 group from left to right, and Figure B shows the immunofluorescence results and cell counting results of astrocytes in the MDL-1 group from left to right. DETAILED DESCRIPTION

[0020] The present invention provides application of MDL-1 in preparing a brain protection drug after ischemic stroke.

[0021] The present invention does not specifically limit the specific source of MDL-1, and any conventional commercially available product in the art can be used. In the present invention, the MDL-1 is preferably able to significantly reduce the apoptosis rate of cells on the infarct side, can significantly inhibit the production of proinflammatory factors, can significantly promote the production of anti-inflammatory factors, and can significantly inhibit the production of inflammatory chemokines. The proinflammatory factors preferably include TNF-α, IL-1β and IL-6, the anti-inflammatory factors preferably include IL-10 and Arg-1, and the inflammatory chemokines preferably include CCL-2, CCL-3, CCL-5 and CXCL-10. In the present invention, the MDL-1 preferably inhibits the activation of microglia and astrocytes, thereby playing a role in regulating post-stroke neuroinflammation.

[0022] The present invention also provides a brain protection drug after ischemic stroke, wherein the active ingredient of the drug includes MDL-1.

[0023] The present invention has no special limitation on other auxiliary materials contained in the drug, and common auxiliary materials in the field of drugs can be used. In the present invention, the drug is preferably an injection preparation, the concentration of MDL-1 in the injection preparation is preferably 0.25μg / ml-0.5μg / ml, and the injection dose of the injection preparation is preferably 1ml per kg body weight.

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Example 1

[0028] The purchased mouse recombinant protein MDL-1 (#8438-CL, R&D Systems, USA) was dissolved in PBS solution at a concentration of 100 μg / mL and stored at -80°C. The MDL-1 solution was diluted to 0.25 μg / ml, 0.5 μg / ml, and 0.75 μg / ml using PBS solution.

[0029] The specific method for constructing a mouse model of unilateral middle cerebral artery occlusion is as follows:

[0030] Adult male C57BL / 6J mice (6-8 weeks old, 20±2g) were selected and anesthetized and intraperitoneally injected with 0.25μg / ml, 0.5μg / ml, and 0.75μg / ml MDL-1 solutions, respectively. The injection volume was approximately 1 ml per kg body weight of the mouse. At the same time, mice that were not injected with the solution were set as the control group. One hour later, a mouse model of unilateral middle cerebral artery occlusion was established.

[0031] First, the left common carotid artery was separated under a dissecting microscope and ligated below the common carotid artery. The artery was separated toward the head to the bifurcation of the internal and external carotid arteries, and the external carotid artery was ligated. A small incision was made at the distal end of the common carotid artery ligature, and a thread plug (diameter: 0.21±0.01mm, Tianjin Minxue Technology, China) was introduced into the internal carotid artery through the small incision. The thread plug was continued to be introduced inward and downward, and stopped when slight resistance was felt. Laser Doppler was used to detect the blood flow of the cerebral cortex on the operated side. After 60 minutes, the thread plug was pulled out to allow reperfusion of blood flow in the cerebral cortex on the operated side.

[0032] Behavioral experiment: The behavioral experiment was completed one day after the mouse model was established, and mNSS scores were performed to evaluate the movement, sensation, and balance functions of the mice. Figure 1 As shown in A. The mice in the control group showed obvious neurological deficits, while the neurological deficits in the mice in the MDL-1 group were milder, and when the MDL-1 concentration was 0.5 μg / ml, the neurological deficits of the mice were the mildest (p=0.0185); when the MDL-1 concentration was 0.75 μg / ml, the neurological deficits of the mice were more severe than those in the control group, which may be due to the neurotoxic effect of MDL-1.

[0033] Example 2

[0034] Example 1 Three days after the mouse model was established, the brain was removed after perfusion with 4°C PBS solution, stained with TTC (2,3,5-triphenyltetrazolium chloride), fixed with paraformaldehyde and photographed. The results are as follows: Figure 1 As shown in B, ImageJ was used to measure the volume of cerebral infarction. Figure 1 C. Figure 1 C It can be seen that, consistent with the improved neurological deficit symptoms, when the MDL-1 concentration was 0.5 μg / ml, the relative cerebral infarction volume measured by TTC staining in the MDL-1 group was significantly smaller than that in the control group (p=0.0276).

[0035] Example 3

[0036] Example 1 Three days after the mice were modeled, they were perfused with 4°C PBS solution and the affected side brain tissue of the group with MDL-1 concentration of 0.5 μg / ml was obtained. Single cell suspension was prepared after grinding, and cell apoptosis was detected by flow cytometry using ANNEXIN V-FITC / PI apoptosis detection kit (#CA1020, Solarbio, China). The results are shown in Figure 1 D and Figure 1 As shown in E, the apoptosis rate of cells on the infarct side in the MDL-1 group was significantly lower than that in the control group (p=0.0381).

[0037] Example 4

[0038] Example 1 Three days after mouse modeling, the affected side brain tissue of the MDL-1 concentration of 0.5 μg / ml group was perfused with 4°C PBS solution, and total RNA was extracted using the SteadyPure universal RNA extraction kit (#AG21017, Acre Bio, China), and reverse transcription was performed using the EvoM-MLV reverse transcription premix kit (#AG11728, Acre Bio, China). The SYBR method Pro TaqHS premix qPCR kit (#AG11718, Acre Bio, China) was used to detect the levels of inflammatory factors (TNF-α, IL-1β, IL-6, IL-10, Arg-1) and chemokines (CCL2, CCL3, CCL5, CXCL10) mRNA by the ABI QuantStudio5 real-time quantitative polymerase chain reaction system. The primer sequences involved are shown in Table 1, and the PCR reaction system and reaction procedure are shown in Table 2 and Table 3, respectively.

[0039] Table 1 Primer sequences

[0040]

[0041] Table 2 Reaction system

[0042]

[0043] Table 3 Reaction procedure

[0044]

[0045] The results are as follows Figure 2 As shown. The mRNA levels of proinflammatory factors TNF-α, IL-1β, and IL-6 in the MDL-1 group were significantly lower than those in the control group ( Figure 2 A). The mRNA levels of anti-inflammatory factors IL-10 and Arg-1 increased significantly ( Figure 2 B). The mRNA levels of chemokines CCL-2, CCL-3, CCL-5 and CXCL-10 were significantly decreased ( Figure 2C). The above results indicate that MDL-1 regulates post-stroke neuroinflammation from pro-inflammatory to anti-inflammatory and inhibits leukocyte infiltration.

[0046] Example 5

[0047] Example 1 Three days after mouse modeling, the affected side of the brain of the group with an MDL-1 concentration of 0.5 μg / ml was obtained after perfusion with 4°C PBS solution, and frozen sections were made after gradient dehydration with sucrose solution. Microglia (A) and astrocytes (B) were labeled with Iba-1 (red) (1:500; #019-19741, Wako, Japan) and GFAP (red) (1:500; #ab7260, Abcam, UK) antibodies, respectively. The number and morphological changes of activated microglia and reactive astrocytes were observed under a fluorescence microscope, and the number of activated microglia and reactive astrocytes was counted using ImageJ.

[0048] The results are as follows Figure 3 As shown in the figure, the total number of microglia and astrocytes in the MDL-1 group was reduced compared with that in the control group. Microglia with shorter processes, cell body diameter greater than 5 μm, and amoeba-like morphology were considered activated microglia, and astrocytes with longer processes and more branches were considered reactive astrocytes. After 40-fold magnification, the number of activated microglia in the MDL-1 group was significantly lower than that in the control group ( Figure 3 A, p = 0.0012). Similarly, the number of reactive astrocytes in the MDL-1 group also showed a decreasing trend ( Figure 3 B, p=0.0370). This indicates that MDL-1 regulates post-stroke neuroinflammation by inhibiting the activation of microglia and astrocytes.

[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of MDL-1 in the preparation of brain protective drugs after ischemic stroke.

2. The use according to claim 1, characterized in that: The MDL-1 can significantly reduce the apoptosis rate of cells on the infarcted side.

3. The use according to claim 1, characterized in that: The MDL-1 can significantly inhibit the production of pro-inflammatory factors, including TNF-α, IL-1β and IL-6.

4. The use according to claim 1, characterized in that: The MDL-1 can significantly promote the production of anti-inflammatory factors, including IL-10 and Arg-1.

5. The use according to claim 1, characterized in that: The MDL-1 can significantly inhibit the production of inflammatory chemokines, including CCL-2, CCL-3, CCL-5 and CXCL-10.

6. The use according to claim 1, characterized in that: The MDL-1 regulates post-stroke neuroinflammation by inhibiting the activation of microglia and astrocytes.

7. A brain protection drug after ischemic stroke, characterized in that: The active ingredients of the drug include MDL-1; The medicine is an injection preparation; The concentration of MDL-1 in the injection preparation is 0.25 μg / ml-0.5 μg / ml.

8. The drug according to claim 7, characterized in that The injection dosage of the injection preparation is 1 ml per kg body weight.

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