Application of HK2 in preparation of hematoma removing medicine for treating cerebral hemorrhage

Abstract

The invention provides application of HK2 in preparation of hematoma removing medicine for treating cerebral hemorrhage. Experiments prove that HK2 enhances the phagocytic function of microglial cells by regulating and controlling a PPARgamma / CD36 signal channel, so that hematoma removal is promoted, the neurological function is improved, and the HK2 has important significance.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of HK2 in preparing a drug for treating hematoma removal after cerebral hemorrhage. Background Art

[0002] Intracerebral hemorrhage (ICH) is a non-traumatic rupture of small arteries in the brain. In China, ICH accounts for 19% to 48% of strokes, with an early mortality rate of 30% to 40%. At the end of 6 months, about 80% of surviving patients still have residual disabilities, which is one of the main causes of death and disability in patients (Wang Longde, Peng Bin, Zhang Hongqi, et al. Summary of "China Stroke Prevention and Treatment Report 2020" [J]. Chinese Journal of Cerebrovascular Diseases, 2022, 19(2): 136-144.). The mass effect caused by hematoma after ICH and the secondary injury driven by hematoma components are the main causes of brain damage in patients. At present, craniotomy is the main means of treating ICH, but it is subject to many limitations and the efficacy is not significant.

[0003] Endogenous hematoma removal is a new research idea. Studies have shown that within 6 hours after ICH, microglia rapidly migrate to the injured area and start the proliferation program. As an important immune defense line of the central nervous system, these cells play a key role in reducing secondary nerve damage and promoting hematoma removal by efficiently phagocytizing hematoma decomposition products, necrotic neuronal fragments and cell debris. The research on microglial hematoma removal after ICH is currently focused on scavenger receptors that are activated by binding to ligands on damaged red blood cells or free heme to prompt cells to ingest and phagocytose them. Type II scavenger receptor CD36 is expressed on the surface of macrophages, and cells that lack phagocytic ability can acquire phagocytic function after transfection with type I scavenger receptor CD36. At the same time, IL-10 can also regulate CD36, thereby accelerating hematoma removal after ICH. In addition, CD36 is also a key target for PPARγ-mediated downstream gene transcription. Activating PPARγ will increase the expression of CD36 and further enhance the phagocytic function of microglia. This suggests that targeting microglial phagocytosis-related targets is an important way to accelerate hematoma clearance. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides the use of HK2 in the preparation of a drug for treating hematoma removal after cerebral hemorrhage. Experiments have shown that HK2 enhances the phagocytic function of microglia by regulating the PPARγ / CD36 signaling pathway, thereby promoting hematoma removal and improving neurological function.

[0005] The present invention adopts the following technical solution:

[0006] Use of HK2 in the preparation of a drug for hematoma clearance after cerebral hemorrhage.

[0007] The amino acid sequence of HK2 is shown in SEQ ID NO:1, specifically as follows: MRLSDETLLEISKRFRKEMEKGLGATTHPTAAVKMLPTFVRSTPDGTEHGEFLALDLGGTNFRVLWVKVTDNGLQKVEMENQIYAIPEDIMRGSGTQLFDHIAECLANFMDKLHIKDKKLPLGFTFSFPCHQTKLDESFLVSWTKGFKSSGVEGRDVVALIRKAIQRRGDFDIDIVAVVNDTVGTMMTCGYDDHNCEIGLIVGTGSNACYMEEMRHIDMVEGDEGRMCINMEWGAFGDDGSLNDIRTEFDQEIDMGSLNPGKQLFEKMISGMYMGELVRLILVKMAKEELLFGGKLSPELLNTGRFETKDISDIEGEKDGIRKAREVLMRLGLDPTQEDCVATHRICQIVSTRSASLCAATLAAVLQRIKENKGEERLRSTIGVDGSVYKKHPHFAKRLHKTVRRLVPGCDVRFLRSEDGSGKGAAMVTAVAYRLADQHRARQKTLEHLQLSHDQLLEVKRRMKVEMERGLSKETHASAPVKMLPTYVCATPDGTEKGDFLALDLGGTNFRVLLVRVRNGKWGGVEMHNKIYAIPQEVMHGTGDELFDHIVQCIADFLEYMGMKGVSLPLGFTFSFPCQQNSLDESILLKWTKGFKASGCEGEDVVTLLKEAIHRREEFDLDVVAVVNDTVGTMMTCGFEDPHCEVGLIVGTGSNACYMEEMRNVELVEGEEGRMCVNMEWGAFGDNGCLDDFRTEFDVAVDELSLNPGKQRFEKMISGMYLGEIVRNILIDFTKRGLLFRGRISERLKTRGIFETKFLSQIESDCLALLQVRAILQHLGLESTCDDSIIVKEVCTVVARRAAQLCGAGMAAVVDRIRENRGLDALKVTVGVDGTLYKLHPHFAKVMHETVKDLAPKCDVSFLQSEDGSGKGAALITAVACRIREAGQR。

[0008] According to one embodiment of the present invention, the application is to enhance the phagocytic function of microglia by regulating the PPARγ / CD36 signaling pathway.

[0009] According to one embodiment of the present invention, the application is to regulate the PPARγ / CD36 signaling pathway in a non-metabolism-dependent manner.

[0010] According to one embodiment of the present invention, the hematoma removal is to reduce the volume of the hematoma.

[0011] According to one embodiment of the present invention, the application is to alleviate secondary brain damage.

[0012] According to one embodiment of the present invention, the application is to promote recovery of neurological function.

[0013] The present invention discloses the use of HK2 in regulating microglial phagocytosis of red blood cells and hematoma clearance after intracerebral hemorrhage (ICH). The present invention has the following significant beneficial effects:

[0014] 1. Promote hematoma removal and reduce secondary brain damage

[0015] After cerebral hemorrhage, the formation of hematoma not only directly damages brain tissue, but also causes secondary brain damage through red blood cell decomposition products (such as hemoglobin, iron ions, etc.). The present invention has proved through experiments that HK2 can significantly enhance the phagocytic ability of microglia to red blood cells and their decomposition products, thereby accelerating the removal of hematoma. This effect effectively reduces the compression and toxic effects of hematoma on surrounding brain tissue, alleviates secondary brain damage, and improves the pathological process after cerebral hemorrhage.

[0016] 2. Improve neurological function and reduce disability rate

[0017] After cerebral hemorrhage, neurological deficits are the main cause of the high disability rate of patients. The present invention proves through behavioral experiments (such as modified Garcia score and Morris water maze test) that HK2 inhibition will aggravate neurological deficits, while HK2 overexpression significantly improves neurological function. This shows that HK2 can effectively promote the recovery of neurological function and reduce the disability rate of patients with cerebral hemorrhage by regulating the function of microglia.

[0018] 3. Enhance phagocytic function through PPARγ / CD36 signaling pathway

[0019] The present invention discloses the mechanism by which HK2 enhances the phagocytic function of microglia by regulating the PPARγ / CD36 signaling pathway. PPARγ is an important transcription factor that can regulate the expression of a variety of genes related to phagocytic function, while CD36 is a scavenger receptor that is directly involved in the recognition and phagocytosis of red blood cells. HK2 significantly enhances the phagocytic ability of microglia by upregulating the expression of PPARγ and CD36, thereby promoting the clearance of hematomas.

[0020] 4. Provide new drug targets for the treatment of cerebral hemorrhage

[0021] At present, there are limited treatments for cerebral hemorrhage, especially for the treatment strategies of hematoma removal and secondary brain injury, which are in urgent need of breakthroughs. The present invention provides a new drug target for the treatment of cerebral hemorrhage by revealing the key role of HK2 in hematoma removal and neurological function recovery. By developing drugs or gene therapy strategies targeting HK2, it is possible to effectively promote hematoma removal, reduce secondary brain injury, and improve the patient's neurological prognosis, which has broad clinical application prospects.

[0022] In summary, the present invention provides a new theoretical basis and drug target for the treatment of cerebral hemorrhage by revealing the key role of HK2 in hematoma removal and neurological function recovery after cerebral hemorrhage. By regulating HK2 and its downstream signaling pathways, it can effectively promote hematoma removal, reduce secondary brain damage, and improve the patient's neurological prognosis, which has important scientific significance and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram showing the expression of HK2 in the tissue surrounding the hematoma after ICH in mice;

[0024] Figure 2 This is a statistical graph showing the effect of HK2 inhibition on neurological function scores after ICH in mice;

[0025] Figure 3 This is a specimen diagram showing the effect of HK2 inhibition on hematoma absorption after ICH in mice;

[0026] Figure 4 This is a statistical graph showing the effect of HK2 inhibition on hematoma absorption after ICH in mice;

[0027] Figure 5 This is the result diagram of the effect of HK2 on the protein expression level of key genes in mouse microglial BV2 cells;

[0028] Figure 6 This is an immunofluorescence image showing the effect of HK2 on the phagocytic ability of red blood cells in mouse microglial BV2 cells. DETAILED DESCRIPTION

[0029] In order to further illustrate the present invention and its advantages, the technical scheme of the present invention is further illustrated by specific embodiments below, and it should be understood that these embodiments only help to understand the present invention and should not be regarded as specific limitations of the present invention. The raw materials and reagents used in the present invention are all commercially available products. Unless otherwise specified, the parts described in the present invention are all weight parts, and the percentages are all mass percentages.

[0030] Example 1

[0031] Western blot was used to investigate the changes in the protein expression level of HK2 in the brain tissue around the hematoma after intracerebral hemorrhage in mice:

[0032] (1) The mouse ICH model was established by injecting collagenase into the basal ganglia of mice (C57 / BL6). The brain tissue around the hematoma was collected at 6 hours, 12 hours, 24 hours, 72 hours, and 7 days after modeling;

[0033] (2) Animal protein extraction: The brain tissues were divided into six groups: sham group (sham), 6-hour group (6h), 12-hour group (12h), 24-hour group (24h), 72-hour group (72h), and 7-day group (7d); the brain tissues were placed in 1.5 ml EP tubes, and 50 mM protease inhibitors and RIPA lysis buffer were prepared at a ratio of 1:50. The lysis buffer was added to each EP tube in proportion (750 ul lysis buffer was added to every 1 g brain tissue), ground with a tissue grinder, sonicated with a cell ultrasonic pulverizer, and then placed on ice to continue lysis for 30 min;

[0034] (4) Centrifuge in a low-temperature high-speed centrifuge at 4°C, 12,000 rpm for 15 min, and collect the protein supernatant;

[0035] (5) Add 1 / 4 volume of 5X SDS-PAGE Sample Loading Buffer to the above 1.5 ml EP tube. After thoroughly mixing, place the sample in a preheated 100°C water bath and suspend and heat for 10 min to fully denature the protein.

[0036] (6) Cool the protein sample on ice and centrifuge it before performing a Western blot experiment or store it in a -80°C refrigerator for a long time for future use;

[0037] (7) Each experiment was repeated 6 times and the Western blot results were quantitatively analyzed.

[0038] The results are as follows Figure 1 As shown, HK2 was significantly upregulated after intracerebral hemorrhage in mice, reaching the highest level at 72 hours.

[0039] Experimental Example 2

[0040] Corner Turn Test was used to investigate the effect of HK2 on neurological function in mice after cerebral hemorrhage:

[0041] (1) Mice 3 days after ICH were divided into four groups: sham group, ICH group, control group (ICH+Vehicle), and HK2 inhibition group (ICH+3-BP). The mice were placed in an environment with no restrictions on movement. The mice were allowed to freely approach a 30° corner, and the number of times the mice freely turned left or right to leave the corner was recorded.

[0042] (2) There was a 1-minute interval between two experiments for each mouse, and a total of 10 experiments were performed. The percentage of mice turning left was then calculated.

[0043] The results are as follows Figure 2 As shown, HK2 plays a role in the recovery of neurological function after intracerebral hemorrhage in mice.

[0044] Experimental Example 3

[0045] The effect of HK2 on the hematoma absorption rate after intracerebral hemorrhage in mice was investigated by calculating the hematoma area:

[0046] (1) The mouse ICH model was established by injecting collagenase into the basal ganglia of mice (C57 / BL6). The mice were divided into two groups according to whether the HK2 inhibitor 3-BP was injected: ICH group and ICH+3-BP group. Brain tissues were collected 1 day, 3 days, and 7 days after modeling.

[0047] (2) The brain tissue was dehydrated and cut into 1 mm brain slices. The brain slices including the hematoma were photographed in sequence. Figure 3 );

[0048] (3) Process the brain slice images, calculate the hematoma volume, and statistically analyze the results ( Figure 4 );

[0049] The results are as follows Figure 3 , Figure 4 As shown, HK2 plays a role in hematoma absorption after intracerebral hemorrhage in mice.

[0050] Example 4

[0051] Western blot was used to investigate the regulatory ability of HK2 on the protein expression levels of two key genes in BV2 cells:

[0052] (1) Untreated BV2 cells (NC group) and BV2 cells overexpressing HK2 via lentivirus (HK2-OE group) in the logarithmic growth phase were digested, centrifuged, resuspended, and then seeded in six-well plates;

[0053] (2) After 24 hours, the six-well plate was removed from the incubator and the cell density and morphology were observed under a microscope to determine whether they were suitable for protein extraction;

[0054] (3) Cell protein extraction: discard the culture medium, wash with 1X PBS washing solution at 4°C for 3 times, aspirate the residual PBS washing solution as much as possible, prepare 50mM protease inhibitor and RIPA lysis solution at a ratio of 1:50, add 120μl lysis solution to each 6-well plate sample well, place on ice to continue lysis, and after 30 minutes, scrape the cells in the 6-well plate with a cell scraper and transfer them to a 1.5ml EP tube;

[0055] (4) Centrifuge in a low-temperature high-speed centrifuge at 4°C, 12,000 rpm for 15 min, and collect the protein supernatant;

[0056] (5) Add 1 / 4 volume of 5X SDS-PAGE Sample Loading Buffer to the above 1.5 ml EP tube. After thoroughly mixing, place the sample in a preheated 100°C water bath and suspend and heat for 10 min to fully denature the protein.

[0057] (6) Cool the protein sample on ice and centrifuge it before performing a Western blot experiment or store it in a -80°C refrigerator for a long time for future use;

[0058] (7) Each group of experiments was repeated 3 times and the Western blot results were quantitatively analyzed ( Figure 5 ).

[0059] The results are as follows Figure 5 As shown, HK2 significantly upregulated the protein expression levels of two key genes (PPARγ, CD36) in BV2 cells.

[0060] Example 5

[0061] The effect of HK2 on the phagocytic ability of microglia was verified by in vitro phagocytosis experiments.

[0062] 1. Mouse erythrocyte extraction: Mouse erythrocytes were extracted using mouse erythrocyte separation solution (TBD, RBC2014MK, China). 200 μL of purified erythrocyte solution was diluted and incubated with carboxyfluorescein succinimidyl ester (CFSE, Selleck, S8269, USA) at 37°C in the dark for 30 minutes. The reaction was stopped by adding ice-cold serum, and the cells were used for in vitro phagocytosis experiments.

[0063] 2. In vitro erythrocyte phagocytosis: BV2 cells were divided into two groups, the control group without special treatment and the HK2-OE group with HK2 overexpressed by lentivirus, and incubated with CFSE-labeled erythrocytes (erythrocyte: phagocytic cell ratio = 10:1) for up to 2 hours. Unphagocytosed free erythrocytes were removed by washing with PBS. Cells were fixed with paraformaldehyde for immunofluorescence staining. The phagocytic index was calculated as the percentage of microglia containing erythrocytes.

[0064] The results are as follows Figure 6 As shown, HK2 can significantly enhance the phagocytic ability of microglia.

[0065] The present invention uses collagenase induction method to establish mouse ICH model, uses HK2 inhibitor 3-BP and PPARγ inhibitor T0070907 for intervention, evaluates neurological function by modified Garcia score and corner test, evaluates hematoma clearance by hemoglobin content determination and immunofluorescence staining, uses BV2 cells to perform in vitro phagocytosis experiment, detects the effect of HK2 on microglial phagocytosis, and detects the expression of PPARγ / CD36 signaling pathway related proteins and genes by Western blot and qPCR. The experimental results show that HK2 expression is significantly increased 72 hours after ICH, HK2 inhibition aggravates neurological deficits and delays hematoma clearance, while HK2 overexpression enhances microglial phagocytosis, HK2 regulates microglial phagocytosis through PPARγ / CD36 signaling pathway, and the regulatory effect is independent of metabolic pathways.

Claims

1. Application of HK2 in the preparation of drugs for the treatment of hematoma removal after cerebral hemorrhage. The amino acid sequence of HK2 is as follows:

2. The use according to claim 1, characterized in that The application is to enhance the phagocytic function of microglia by regulating the PPARγ / CD36 signaling pathway.

3. The use according to claim 1, characterized in that The application is to regulate the PPARγ / CD36 signaling pathway in a non-metabolism-dependent manner.

4. The use according to any one of claims 1 to 3, characterized in that: The hematoma removal is to reduce the hematoma volume.

5. The use according to any one of claims 1 to 3, characterized in that: The application is to alleviate secondary brain damage.

6. The use according to any one of claims 1 to 3, characterized in that: The application is to promote the recovery of nerve function.

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