Use of del-1 protein in the preparation of a medicament for treating secondary injury following traumatic brain injury

Abstract

The application provides application of Del-1 protein in preparation of a drug for treating secondary injury after craniocerebral trauma, and belongs to the technical field of medical uses. The Del-1 protein can not only remove pathological microvesicles in peripheral blood to prevent and treat coagulopathy after TBI (traumatic brain injury), but also stabilize vascular endothelial cells, so that the Del-1 protein can prevent blood brain barrier leakage and brain edema after TBI. Moreover, the Del-1 protein is a kind of protein normally secreted in human body, and has good safety and practicability. The Del-1 protein can be used for preparing a drug for treating secondary injury after trauma. The Del-1 protein can be obtained from human endothelial cells or expressed in a large amount through in-vitro cell culture. Moreover, the protein can also stabilize endothelial cells, and has more remarkable function in treating endothelial disorders.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical applications, and in particular to the use of Del-1 protein in the preparation of drugs for treating secondary injuries following traumatic brain injury. Background Technology

[0002] Del-1 is a 52 kDa glycoprotein containing a PS-binding C1C2 domain and an integrin-binding RGD finger, thus allowing it to clear PS+EVs like MFG-E8. Del-1 shares over 50% sequence similarity with MFG-E8, exhibiting highly similar protein structures. Furthermore, Del-1 is synthesized by endothelial cells and plays a crucial role in endothelial homeostasis, offering better protection against TBI-induced endothelial lesions. Previous studies have shown that intravenous injection of Del-1 promotes angiogenesis and muscle function recovery in limb ischemia-inducing mice and improves endothelial function in mice with cardiovascular disease. In LPS-induced pneumonia, Del-1-deficient mice showed increased neutrophil accumulation on endothelial cells, and in an autoimmune encephalomyelitis model, increased blood-brain barrier (BBB) ​​disruption. However, there are no reports on the therapeutic effects of Del-1 protein on secondary injuries following traumatic brain injury. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides the application of Del-1 protein in the preparation of drugs for treating secondary injuries following traumatic brain injury, thus providing a new use for Del-1 protein.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] This invention provides the application of Del-1 protein in the preparation of drugs for treating secondary injuries following traumatic brain injury.

[0006] Preferably, the amino acid sequence of the Del-1 protein is shown in SEQ ID No. 1.

[0007] This invention also provides the application of Del-1 protein in the preparation of drugs for clearing microvesicles after traumatic brain injury.

[0008] Preferably, the microvesicles include one or more of mitochondrial microvesicles, brain-derived microvesicles, endothelial cell-derived microvesicles, and platelet-derived microvesicles.

[0009] This invention also provides the application of Del-1 protein in the preparation of drugs for the prevention and treatment of coagulopathy after traumatic brain injury.

[0010] This invention also provides the application of Del-1 protein in the preparation of drugs for preventing blood-brain barrier leakage and cerebral edema after traumatic brain injury.

[0011] The beneficial effects of this invention are as follows:

[0012] In the treatment of traumatic brain injury (TBI), Del-1 protein not only clears pathological microvesicles in peripheral blood to prevent coagulopathy after TBI, but also stabilizes vascular endothelial cells, thus preventing blood-brain barrier leakage and cerebral edema after TBI. Furthermore, because Del-1 protein is a normally secreted protein in the human body, its safety and applicability are good. It can be used to prepare drugs for treating secondary injuries after trauma. Del-1 protein can be obtained from human endothelial cells or expressed in large quantities through in vitro cell culture. Moreover, this protein can stabilize endothelial cell function, making its function in treating endothelial disorders even more significant. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0014] Figure 1 To explore whether Del-1 can promote microvesicle clearance by measuring the number of microvesicles in the plasma of TBI mice after pre-injection of Del-1 protein.

[0015] Figure 2 To explore the coagulation status of plasma in mice that have undergone TBI by pre-injecting Del-1 protein, and to observe whether Del-1 can prevent coagulopathy after TBI.

[0016] Figure 3 To investigate whether Del-1 can prevent vascular endothelial barrier damage after TBI by measuring Evans blue leakage in brain tissue after pre-injection of Del-1 protein in mice. Detailed Implementation

[0017] This invention provides the application of Del-1 protein in the preparation of drugs for treating secondary injuries following traumatic brain injury. This invention does not specifically limit the dosage form or preparation method of the drug; a medically acceptable dosage form of Del-1 protein can be used, and it can be prepared using conventional methods. In this invention, the Del-1 protein is a 52 kDa endothelial cell secretory glycoprotein, also identified as a microvesicle clearance protein. Del-1 contains three n-terminal epidermal growth factor (EGF)-like repeats and two c-terminal discoid protein-like domains, and is therefore also referred to as EDIL3 (EGF-like repeats and discoid protein-like domain 3). In this invention, the amino acid sequence of the Del-1 protein is shown in SEQ ID No. 1, specifically as follows:

[0018] MKRSVAVWLLVGLSLGVPQFGKGDICDPNPCENGGICLPGLADGSFSCECPDGFTDPNCSSVVEVASDEEEPTSAGPCTPNPCHNGGTCEISEAYRGDTFIGYVCKCPRGFNGIHCQHNI NECEVEPCKNGGICTDLVANYSCECPGEFMGRNCQYKCSGPLGIEGGIISNQQITASSTHRALFGLQKWYPYYARLNKKGLINAWTAAENDRWPWIQINLQRKMRVTGVITQGAKRIGSP EYIKSYKIAYSNDGKTWAMYKVKGTNEDMVFRGNIDNNTPYANSFTPPIKAQYVRLYPQVCRRHCTLRMELLGCELSGCSEPLGMKSGHIQDYQITASSIFRTLNMDMFTWEPRKARLDK QGKVNAWTSGHNDQSQWLQVDLLVPTKVTGIITQGAKDFGHVQFVGSYKLAYSNDGEHWTVYQDEKQRKDKVFQGNFDNDTHRKNVIDPPIYARHIRILPWSWYGRITLRSELLGCTEEE.

[0019] This invention also provides the application of Del-1 protein in the preparation of drugs for clearing microvesicles after traumatic brain injury. Preferably, the microvesicles include one or more of mitochondrial microvesicles, brain-derived microvesicles, endothelial cell-derived microvesicles, and platelet-derived microvesicles. This invention does not impose any particular limitations on the dosage form or preparation method of the drug; a medically acceptable dosage form of Del-1 protein can be used, and the drug can be prepared using conventional methods.

[0020] This invention also provides the application of Del-1 protein in the preparation of drugs for the prevention and treatment of coagulopathy after traumatic brain injury. This invention does not impose any particular limitations on the dosage form or preparation method of the drug; a medically acceptable dosage form of Del-1 protein can be used, and the drug can be prepared using conventional methods.

[0021] This invention also provides the application of Del-1 protein in the preparation of drugs for preventing blood-brain barrier leakage and cerebral edema after traumatic brain injury. This invention does not impose any particular limitations on the dosage form or preparation method of the drug; a medically acceptable dosage form of Del-1 protein can be used, and the drug can be prepared using conventional methods.

[0022] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1

[0024] 1. Effect of Del-1 on microvesicle content after TBI in mice

[0025] Experimental groups: sham operation group + PBS group; FPI + PBS group; FPI + Del-1 group (PBS: phosphate buffer, pH=7.4)

[0026] Sham surgery group: Mice only had bone windows opened, but no hydraulic impact was performed;

[0027] FPI: fluid percussion injury pressure is 1.9 ± 0.1 atm;

[0028] The Del-1 group used 400 mg / kg / animal, totaling 100 μl, while the PBS group used the same volume of PBS.

[0029] Experimental Procedure: Twenty-four mice were randomly divided into three groups as described above. In the sham-operated group + PBS group, only a bone window was opened. Before opening the bone window, 100 μL of sterile PBS was injected into the tail vein (total 100 μL). In the FPI + PBS group, a bone window was opened, and TBI was induced using a hydraulic impaction model. Before modeling, 100 μL of sterile PBS was injected into the tail vein. In the FPI + Del-1 group, a bone window was opened, and TBI was induced using a hydraulic impaction model. Before modeling, Del-1 protein (400 mg / kg / mouse, total 100 μL) was injected into the tail vein. Blood was collected 3 hours after modeling, and platelet-rich plasma was obtained by centrifugation. Microparticle labeling was performed using mitotrackergreen (mitochondrial microvesicles), anti-GFAP antibody (brain-derived microvesicles), anti-CD31 antibody (endothelial cell-derived microvesicles), and anti-CD41 antibody (platelet-derived microvesicles), respectively. The positive rates of each indicator were detected by flow cytometry. Results are shown below. Figure 1 .

[0030] Experimental Results: Compared with the sham-operated + PBS group, the positive rates of mitotrackergreen, anti-GFAP antibody, anti-CD31 antibody, and anti-CD41 antibody in the FPI+PBS group were significantly increased, from 3.81%±2.03% to 16.31%±3.32%, from 0.81%±0.48% to 3.81%±1.58%, from 0.74%±0.46% to 2.66%±0.88%, and from 1.61%±0.90% to 8.89%±1.50%, respectively. Compared with the FPI+PBS group, the positive rates of mitotrackergreen, anti-GFAP antibody, anti-CD31 antibody, and anti-CD41 antibody in the FPI+Del-1 group were significantly decreased, to 10.23%±1.33%, 2.16%±1.22%, 0.83%±0.64%, and 3.87%±1.33%, respectively.

[0031] Experimental conclusion: Del-1 protein can clear mitochondrial microvesicles, brain-derived microvesicles, endothelial cell-derived microvesicles and platelet-derived microvesicles released after TBI.

[0032] 2. The therapeutic effect of Del-1 on hypercoagulable state of blood after TBI in mice.

[0033] Experimental groups: sham surgery group + PBS group; FPI + PBS group; FPI + Del-1 group, same as in Example 1.

[0034] Experimental Procedure: Twenty-four mice were randomly divided into three groups as described above. In the sham-operated group + PBS group, only a bone window was opened. Before opening the bone window, 100 μl of sterile PBS was injected into the tail vein. In the FPI + PBS group, a bone window was opened, and TBI was induced using a hydraulic impact model. Before modeling, 100 μl of sterile PBS was injected into the tail vein. In the FPI + Del-1 group, a bone window was opened, and TBI was induced using a hydraulic impact model. Before modeling, 100 μl of Del-1 protein (400 mg / kg / mouse) was injected into the tail vein. Blood samples were collected at 3 h and 6 h after modeling, and platelet-rich plasma was obtained by centrifugation. Prothrombin time and activated partial thromboplastin time were measured. Results are shown below. Figure 2 .

[0035] Experimental Results: Compared with the sham-operated group + PBS, the FPI + PBS group showed significantly higher levels of hypercoagulability at 3h and 6h post-modeling. Specifically, prothrombin time (PT) decreased from 15.17s ± 2.1s to 11.5s ± 1.34s at 3h and from 15.89s ± 1.89s to 10.7s ± 1.28s at 6h. Activated partial thromboplastin time (APTT) decreased from 29.94s ± 3.28s to 22.15s ± 2.3s at 3h and from 28.7s ± 2.93s to 21.3s ± 2.38s at 6h. Compared with the FPI+PBS group, the hypercoagulable state in the FPI+Del-1 group was significantly reversed. Specifically, prothrombin time (PT) recovered to 14.56s±1.72s and 14.6s±2.13s at 3h and 6h, respectively, and activated partial thromboplastin time (APTT) recovered to 27.7s±2.01s and 27.2s±1.08s at 3h and 6h, respectively.

[0036] Experimental conclusion: Del-1 can reverse the hypercoagulable state in mice after TBI.

[0037] 3. Effects of Del-1 on blood-brain barrier disruption after TBI in mice

[0038] Experimental groups: sham surgery group + PBS group; FPI + PBS group; FPI + Del-1 group, same as in Example 1.

[0039] Experimental Procedure: Twenty-four mice were randomly divided into three groups as described above. In the sham-operated group + PBS group, bone windows were opened, and 100 μl of sterile PBS was injected into the tail vein before opening the bone window. In the FPI + PBS group, bone windows were opened, and TBI was induced using a hydraulic impaction model, with 100 μl of sterile PBS injected into the tail vein before modeling. In the FPI + Del-1 group, bone windows were opened, and TBI was induced using a hydraulic impaction model, with 100 μl of Del-1 protein (400 mg / kg / mouse) injected into the tail vein before modeling. Four hours after modeling, 100 μl of 2% Evansblue solution was injected into the tail of each mouse. Six hours after modeling, the mice were perfused with PBS, and brain tissue was then harvested. Experimental results are shown below. Figure 3 .

[0040] Experimental results: Compared with the FPI+PBS group, the sham surgery + PBS group showed a significant increase in cerebral endothelial leakage, with Evans Blue leakage density increasing from 2.97 ODU ± 0.44 ODU to 12.63 ODU ± 3.07 ODU. Compared with the FPI+PBS group, the cerebral endothelial leakage Evans Blue in the FPI+Del-1 group was significantly reversed, with Evans Blue leakage density decreasing to 7.17 ODU ± 1.75 ODU.

[0041] Experimental conclusion: Del-1 can reverse blood-brain barrier disruption in mice after TBI.

[0042] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of Del-1 protein in the preparation of drugs for the prevention and treatment of secondary injuries following traumatic brain injury, characterized in that, The amino acid sequence of the Del-1 protein is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The Del-1 protein clears pathological microvesicles following traumatic brain injury.

3. The application according to claim 2, characterized in that, The pathological microvesicles are one or more of the following: mitochondrial microvesicles, brain-derived microvesicles, endothelial cell-derived microvesicles, and platelet-derived microvesicles.

4. The application according to claim 1, characterized in that, The Del-1 protein prevents coagulopathy after traumatic brain injury.

5. The application according to claim 1, characterized in that, The Del-1 protein prevents blood-brain barrier leakage and cerebral edema after traumatic brain injury.

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