Polypeptide Tat-CIRP and application thereof

Abstract

The invention discloses a polypeptide Tat-CIRP and application thereof, the amino acid sequence of the polypeptide Tat-CIRP is as shown in SEQ ID NO.3, Tat is a transmembrane structure sequence of 11 amino acids on a Tat protein, and is specifically as shown in SEQ ID NO.1; cIRP is 14 amino acid sequences on a CIRP protein with the highest affinity with GPR68, and is specifically shown as SEQ ID NO. 2. It is found through experimental research that nerve injury and encephaledema after subarachnoid hemorrhage can be remarkably improved through polypeptide Tat-CIRP administration so as to relieve early brain injury, white matter injury is relieved by promoting myelin sheath repair after subarachnoid hemorrhage, and a complete experimental basis is provided for clinical application of the polypeptide Tat-CIRP.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a polypeptide Tat-CIRP and an application thereof. Background Art

[0002] Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease, usually caused by aneurysm rupture. After bleeding, blood flows into the subarachnoid space, leading to a series of pathological and physiological changes such as increased intracranial pressure, decreased cerebral blood flow, and inflammatory response. Subarachnoid hemorrhage is a complex pathological and physiological process involving multiple genes and signaling pathways. Its basic pathological changes include primary brain damage, early brain damage, and delayed brain damage (Schneider, UC; Xu, R.; Vajkoczy, P. Inflammatory events following subarachnoid hemorrhage (SAH). Curr. Neuropharmacol. 2018, 16, 1385-1395.).

[0003] Primary brain injury is caused by stress injury. Early brain injury (EBI) refers to pathological processes that occur within 72 hours after SAH, including neuroinflammation, microcirculatory dysfunction, blood-brain barrier (BBB) ​​damage, oxidative cascade, brain edema and neuronal cell death, which have been confirmed to be an important factor for poor prognosis of SAH. Delayed brain injury (DBI) refers to the pathological and physiological processes that occur in the late stage of SAH (usually 3-14 days), including brain edema, increased intracranial pressure, acute cerebral vasospasm (CVS), decreased cerebral blood flow, etc.

[0004] Studies have found that early brain injury is closely related to poor prognosis of patients. After aneurysm rupture, primary dysfunction activates the downstream pathways that produce EBI, resulting in pathological changes such as hemorrhagic extravasation, rapid increase in intracranial pressure, acute vasospasm, low cerebral blood flow, cerebrovascular autoregulation dysfunction and brain swelling. After SAH, the blood flow to brain tissue decreases, leading to the accumulation of hemoglobin decomposition products. Brain tissue hypoxia causes changes in local metabolism, brain tissue swelling, and brain tissue acidosis. The mechanisms of subarachnoid hemorrhage are complex and diverse, and there is still a lack of clear understanding of their interrelationships. At present, effective control and reduction of early brain injury (EBI) is the key to treatment. Therefore, finding biomarkers and therapeutic targets related to inflammation in subarachnoid hemorrhage is the focus of related research, which can not only provide more theoretical basis for the diagnosis of the severity and prognosis of subarachnoid hemorrhage, but also provide new diagnosis and treatment methods for patients with subarachnoid hemorrhage.

[0005] After SAH, brain tissue undergoes acidosis, which is caused by the accumulation of hemoglobin breakdown products and changes in local metabolism. As a G protein-coupled receptor sensitive to extracellular acidity and ischemic state, GPR68 may respond to this acidotic environment and participate in the pathological process after SAH. Some studies have shown that the activation of GPR68 may affect brain damage after SAH by regulating intracellular calcium ion concentration, the release of inflammatory mediators, and cell survival and death. GPR68 may participate in the pathological process after intracerebral hemorrhage by regulating intracellular calcium ion concentration and the release of inflammatory mediators. For example, the activation of GPR68 may promote the mobilization of intracellular calcium ions, thereby affecting cell survival and death. In addition, GPR68 may also affect the inflammatory response after intracerebral hemorrhage by regulating the release of inflammatory mediators.

[0006] There is cell apoptosis and necrosis after subarachnoid hemorrhage. After cell apoptosis or necrosis, a large number of cell signaling molecules are released, which can trigger inflammation and are called danger-associated molecules, including high mobility groupbox 1 (HMGB1), heat shock proteins, cold shock proteins, etc. CIRP (cold shock protein) is a stress response protein that can be activated by various cell stressors and is exerted by cells according to the specific cell environment, that is, the specific presence of other stress response molecules, and is widely involved in various physiological and pathological processes in the body. CIRP is a new type of inflammatory mediator that can be secreted and released from the heart and liver into the circulatory system in hemorrhagic shock and sepsis, activating the downstream JAK-STAT3 signaling pathway to upregulate the expression of TNF-α and HMGB1, playing the role of damage-associated molecular protein (DAMP) and triggering an inflammatory response. At present, CIRP has been identified as an important mediator in severe inflammation or ischemia, and has the harmful function of aggravating cell damage. At the same time, studies have shown that tumor necrosis factor α (TNF-α), lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) induced GPR68 expression in primary human monocytes and MM6 monocyte cell lines; NF-κB inhibitors blocked these treatment-induced GPR68 expression. Using GPR68 as a new therapeutic target can effectively reduce neuronal damage in ischemia and a variety of other neurological diseases.

[0007] Subarachnoid hemorrhage (SAH) is not only a major clinical problem to be solved, but also a major public health issue. Although progress has been made in the clinical diagnosis and treatment of diseases such as aneurysmal subarachnoid hemorrhage, it is still a disease with extremely high mortality and disability rates, and also brings huge material and human costs. Its key mechanism is still unclear. Therefore, it is urgent to identify feasible therapeutic targets and develop effective drugs in order to provide the best treatment strategy for SAH patients to improve survival rate and prognosis. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides a polypeptide Tat-CIRP and its application. The present invention studies the effect of polypeptide Tat-CIRP on subarachnoid hemorrhage, confirms that the polypeptide has a clear neuroprotective effect on subarachnoid hemorrhage, and provides a relatively complete experimental basis for its clinical application.

[0009] The present invention is achieved through the following technical solutions:

[0010] A polypeptide Tat-CIRP, the amino acid sequence of the polypeptide Tat-CIRP is shown in SEQ ID NO.3, wherein Tat is an 11-amino acid transmembrane structure sequence on the Tat protein, specifically as shown in SEQ ID NO.1; CIRP is a 14-amino acid sequence on the CIRP protein with the highest affinity for GPR68, specifically as shown in SEQ ID NO.2.

[0011] The use of the above-mentioned polypeptide Tat-CIRP in the preparation of drugs for intervention and treatment of cerebrovascular diseases.

[0012] Preferably, the cerebrovascular disease is subarachnoid hemorrhage.

[0013] Preferably, in the polypeptide Tat-CIRP, the Tat amino acid fragment drives the polypeptide Tat-CIRP to penetrate and enter the cell, and the CIRP amino acid fragment can recognize GPR68 and bind to GPR68 to competitively inhibit the formation of the CIRP-GPR68 complex, thereby exerting the neuroprotective effect of GPR68 and further achieving the effect of intervening in the treatment of subarachnoid hemorrhage.

[0014] Preferably, the drug is in any pharmaceutically therapeutically acceptable dosage form.

[0015] A pharmaceutical composition for interventional treatment of cerebrovascular diseases, comprising the above-mentioned polypeptide Tat-CIRP.

[0016] Preferably, a pharmaceutically acceptable drug delivery carrier is also included.

[0017] The beneficial effects of the present invention are as follows:

[0018] The present invention uses a new synthetic polypeptide Tat-CIRP, wherein Tat is a transmembrane structure sequence of 11 amino acids, and the transcriptional transactivator protein Tat can cross the membrane and enter the interior of the cell, and has a strong internalization effect. The polypeptide Tat-CIRP is used to prepare a drug for intervention treatment of subarachnoid hemorrhage, wherein the amino acid fragments in the transcriptional transactivator protein Tat drive the polypeptide Tat-CIRP to penetrate and enter the cell, and the amino acid fragments of the CIRP partial sequence can recognize GPR68 and bind to GPR68 to competitively inhibit the formation of the CIRP-GPR68 complex, thereby exerting the neuroprotective effect of GPR68, and then playing the role of intervention treatment of subarachnoid hemorrhage. Specifically, the administration of the polypeptide Tat-CIRP can significantly improve the nerve damage and brain edema after subarachnoid hemorrhage, thereby alleviating early brain damage, and reduce brain white matter damage by promoting myelin repair after subarachnoid hemorrhage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a study on the improvement of neurological function by the polypeptide Tat-CIRP in Example 2: A is the neurological function score of each group of mice; B is the percentage of brain water content of each group of mice;

[0020] Figure 2 The study of the polypeptide Tat-CIRP on myelin repair in Example 3: A is a whole brain image of MBP immunofluorescence staining of coronal sections after subarachnoid hemorrhage; B is an MBP and SMI-32 immunofluorescence double staining image of the corpus callosum region (selected area in Figure A) of each group of mice, scale = 50μm; C is a statistical graph of SMI-32 / MBP fluorescence intensity in the corpus callosum region of each group of mice, and the data are expressed as mean ± standard error. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0023] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0024] The experimental animals in the following examples are C57BL / 6 mice, provided by the Experimental Animal Center of Nantong University. Adult male C57BL / 6 mice (8-12 weeks old) were used in the experiment, weighing 22-28 g. All animals were kept in light and dark for 12 hours each day, maintaining the normal rhythm of the animals, and free access to food and water. The animal room maintained normal humidity and good ventilation. All animals were first adapted to the breeding environment for 1 week before undergoing surgery, and fasted for 12 hours before surgery, but water was not forbidden.

[0025] The present invention first discovered that GPR68 specifically binds to a segment of CIRP (the amino acid sequence is shown in SEQ ID NO.2), and the free energy of interaction between the two proteins was -17.8 kcal / mol (the lower the free energy, the more stable the structure), which indicates that the two have a strong binding affinity. It is also speculated that CIRP can bind to GPR68 to inhibit the neuroprotective effect of GPR68. This suggests that we can enable GPR68 to exert its neuroprotective effect by inhibiting the binding of CIRP to GPR68 in microglia under inflammatory conditions. Based on the above findings and speculations, the following experiments were designed to verify them.

[0026] Example 1

[0027] A polypeptide Tat-CIRP, whose amino acid sequence is shown in SEQ ID NO.3; wherein Tat is an 11-amino acid transmembrane structure sequence on the Tat protein, specifically as shown in SEQ ID NO.1; CIRP is a 14-amino acid sequence on the CIRP protein with the highest affinity for GPR68, specifically as shown in SEQ ID NO.2.

[0028] The transcriptional transactivator protein Tat can cross the membrane and enter the cell interior, and has a strong internalization effect. The Tat protein is generally composed of 86-102 amino acid residues. The present invention selects the 47-57 amino acid fragment in the Tat protein, and the sequence of the fragment is shown in SEQ ID NO.1, abbreviated as YGRKKRRQRRR, specifically Try-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg. The amino acid fragment is located at the N-terminus of the polypeptide Tat-CIRP.

[0029] The 14 amino acid sequences with the highest affinity between CIRP and GPR68 are the amino acid fragments at positions 147-160 of the CIRP protein. The fragment sequence is shown in SEQ ID NO. 2, abbreviated as QSGGYSDRSSGGSY, specifically Gln-Ser-Gly-Gly-Tyr-Ser-Asp-Arg-Ser-Ser-Gly-Gly-Ser-Tyr.

[0030] Based on this, the amino acid sequence of the polypeptide Tat-CIRP is shown in SEQ ID NO. 3, abbreviated as YGRKKRRQRRRQSGGYSDRSSGGSY, specifically Try-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gln-Ser-Gly-Gly-Tyr-Ser-Asp-Arg-Ser-Ser-Gly-Gly-Ser-Tyr.

[0031] Example 2 Study on the improvement of neurological function by peptide Tat-CIRP

[0032] In order to study the effect of peptide Tat-CIRP on improving neurological function of mice after subarachnoid hemorrhage, and whether it can alleviate early brain damage by improving behavioral manifestations after subarachnoid hemorrhage, we conducted the following experiments using a sham operation group, a control group, and a control and peptide intervention group.

[0033] 1. Group experiment

[0034] The surgical procedure was as follows: an in vivo model of subarachnoid hemorrhage was constructed using the intravascular perforation method. Adult male C57BL / 6 mice were placed in a supine position and the neck was opened with a sharp scalpel in the midline. After locating the appropriate blood vessels, a 4-0 nylon suture was inserted into the left internal carotid artery through the stump of the external carotid artery until resistance was detected. The suture was further advanced 3 mm to perforate the bifurcation of the anterior and middle cerebral arteries and then immediately withdrawn to simulate subarachnoid hemorrhage (SAH) caused by intracranial artery rupture.

[0035] (1) Sham operation group: The above-mentioned surgical procedure was used, and the filament material touched the wall of the blood vessels in the brain but did not penetrate it.

[0036] (2) Control group: The above-mentioned surgical procedure was used to puncture the blood vessel wall to create SAH model.

[0037] (3) Control + peptide intervention group: The above-mentioned surgical procedure was used, and the peptide Tat-CIRP (2 mg / kg) was administered intravenously 2 hours after subarachnoid hemorrhage.

[0038] 2. Observation indicators

[0039] (1) Neurological function score (modified Garcia score) is used to evaluate the degree of brain damage after subarachnoid hemorrhage, including the following: spontaneous activities (0-3 points), bilateral tactile sensation of the body (1-3 points), bilateral whisker touch reaction (0-3 points), climbing movement (1-3 points), forelimb extension function (0-3 points) and symmetry of all limb movements (1-3 points). The score ranges from 3 to 18 points. The larger the value, the milder the neurological damage. 18 points is normal.

[0040] (2) Brain water content determination can be used to measure the degree of brain edema after brain injury. The higher the brain water content ratio, the more severe the edema. Brain edema is an inevitable consequence of cerebral ischemia. The reduction and control of early brain edema can significantly reduce the mortality and disability rates. Therefore, brain water content determination has been widely tested and studied in various brain injury models. Subarachnoid hemorrhage is often accompanied by severe brain damage and brain edema. Therefore, the degree of brain edema in mice can be observed to determine whether the peptide Tat-CIRP can help improve the degree of brain damage after subarachnoid hemorrhage. The specific operation is as follows:

[0041] After the mice were decapitated, the brain was removed within 30 seconds. After the blood stains on the brain surface were absorbed with filter paper, about 100 mg of the left parietal cortex was sharply separated. The wet weight was measured with an electronic analytical balance, and then placed in a 110°C constant temperature drying oven to dry (24 hours), and the dry weight was measured. The percentage of brain water content in each part was calculated using the Elliot formula:

[0042] Brain water content (%) = [(wet weight - dry weight) / wet weight] × ratio

[0043] 3. Experimental results

[0044] like Figure 1 As shown, the neurological function scores of the control + peptide intervention group ( Figure 1 A) and degree of brain edema ( Figure 1 (B) were all improved, and the difference was statistically significant (P<0.01).

[0045] It can be seen from the experimental results of this example that the administration of the polypeptide Tat-CIRP can significantly improve the nerve damage and brain edema after subarachnoid hemorrhage, thereby alleviating early brain damage.

[0046] Example 3 Study on the effect of peptide Tat-CIRP on myelin repair

[0047] This example studies whether the peptide Tat-CIRP can alleviate brain white matter damage by promoting myelin repair after subarachnoid hemorrhage, as follows:

[0048] 1. Group experiment

[0049] This example uses a sham operation group, a control group, and a control and polypeptide intervention group to conduct experiments. The specific grouping is the same as the experimental steps in Example 2.

[0050] 2. Immunofluorescence double labeling method

[0051] The white matter (WM) of the brain is composed of myelin-wrapped axons, oligodendrocytes (OL), astrocytes, and microglia. White matter can control the signals shared by neurons and coordinate the normal functions of brain regions. White matter damage is also involved in the pathological process of early brain damage after subarachnoid hemorrhage. Promoting the recovery of brain white matter helps the recovery of cognitive function. Considering that white matter is related to myelin, we used MBP and SMI-32 immunofluorescence double labeling to evaluate changes in myelin integrity in the corpus callosum. MBP is a marker protein for myelin. SMI-32 is a dephosphorylated neurofilament protein, and its expression often increases after axonal injury. The specific operations are as follows:

[0052] Two days after modeling, the mice were deeply anesthetized, perfused with saline, and then perfused with 4% paraformaldehyde. The mouse brains were dissected and fixed in 4% paraformaldehyde overnight at 4°C, soaked in 20% sucrose in 0.1M phosphate buffer for 1 day, and then soaked in 30% sucrose in 0.1M phosphate buffer for 1 day. After completely descending to the bottom of the 30% sucrose solution, the mouse brain was sliced ​​into 15μm coronal frozen sections using a slicer (Leica CM1950). After rinsing onto a slide, it was washed 3 times (10min each time) with PBST (PBS+0.3% Trition), blocked with 10% donkey serum at room temperature for 1h, and then incubated with the following primary antibodies at 4°C overnight: anti-myelin basic protein (MBP) (1:300 Abcam, USA), anti-neurofilament H (NF-H) (1:300 Biolegend, USA). The appropriate secondary antibody (1:800 Jackson, USA) was selected and incubated with the sections at room temperature for 2 h for staining. The sections were visualized and photographed using a confocal microscope (TCS SP8 Leica, GER). Immunofluorescence staining was evaluated and fluorescence intensity was measured using ImageJ.

[0053] 3. Experimental results

[0054] like Figure 2 As shown in A and B, the expression level of SMI-32 in the control group was significantly increased, while the opposite was true in the control + peptide intervention group. At the same time, compared with the control group, the expression of MBP in the control + peptide intervention group increased. Figure 2 As shown in center C, the fluorescence intensity ratio of SMI-32 / MBP is at a relatively low level.

[0055] The experimental results of this example show that the polypeptide Tat-CIRP can promote myelin repair in mice after subarachnoid hemorrhage, thereby reducing brain white matter damage.

[0056] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A polypeptide Tat-CIRP, characterized in that The amino acid sequence of the polypeptide Tat-CIRP is shown in SEQ ID NO.3, wherein Tat is an 11-amino acid transmembrane structure sequence on the Tat protein, specifically as shown in SEQ ID NO.1; CIRP is a 14-amino acid sequence on the CIRP protein with the highest affinity for GPR68, specifically as shown in SEQ ID NO.

2.

2. Use of the polypeptide Tat-CIRP as claimed in claim 1 in the preparation of drugs for intervention and treatment of cerebrovascular diseases.

3. The use according to claim 2, characterized in that: The cerebrovascular disease is subarachnoid hemorrhage.

4. The use according to claim 3, characterized in that: In the polypeptide Tat-CIRP, the Tat amino acid fragment drives the polypeptide Tat-CIRP to penetrate and enter the cell, and the CIRP amino acid fragment can recognize GPR68 and bind to GPR68 to competitively inhibit the formation of the CIRP-GPR68 complex, thereby exerting the neuroprotective effect of GPR68 and further playing the role of intervention in the treatment of subarachnoid hemorrhage.

5. The use according to claim 2, characterized in that: The drug is in any therapeutically acceptable dosage form.

6. A pharmaceutical composition for interventional treatment of cerebrovascular diseases, characterized in that: It comprises the polypeptide Tat-CIRP as claimed in claim 1.

7. The pharmaceutical composition according to claim 6, characterized in that Also included are pharmaceutically acceptable delivery vehicles.

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