CN112079933A - Polypeptide analogue and application thereof in medicine for intervening and treating cerebral hemorrhage - Google Patents
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Abstract
The invention discloses a polypeptide analogue and application thereof in drugs for intervening and treating cerebral hemorrhage, wherein the polypeptide in the polypeptide analogue is Tat-CIRP-CMA, wherein Tat is a transmembrane structure sequence of 11 amino acids, cold-induced protein CIRP is a 15 amino acid sequence with the highest affinity with MD2, and CMA is a signal peptide sequence of 14 amino acids targeting proteasome.
Description
Technical Field
The invention relates to the technical field of cerebral hemorrhage medicaments, in particular to a polypeptide analogue and application thereof in medicaments for intervening and treating cerebral hemorrhage.
Background
Post-hemorrhagic brain injury is a complex pathophysiological process in which multiple genes and signaling pathways are involved, with the basic pathological changes after cerebral hemorrhage including primary and secondary injuries (Feigin, v.l., Lawes, c.m., Bennett, d.a. and Anderson, c.s. (2003).
The primary injury is mainly mechanical injury caused by tissue destruction during bleeding, hematoma enlargement and intracranial pressure increase. Mechanisms of secondary injury include inflammatory responses, apoptosis, necrosis, red blood cell lysis, and formation of cerebral edema (Xi, g., Keep, r.f. and Hoff, j.t. (2006).
It has been found that inflammation plays an important role in secondary injury after cerebral hemorrhage, and the inflammatory mechanism mainly involves the activation of microglia, infiltration of inflammatory cells and release of cytokines and inflammatory chemokines, which finally leads to cell death and thus aggravates brain injury. The mechanism of cerebral hemorrhage is complex and various, and the correlation is still lack of clear understanding. At present, effective control and alleviation of inflammation secondary injury after cerebral hemorrhage are the key points of cerebral hemorrhage neuroprotection treatment. Therefore, the search for biomarkers and treatment targets related to cerebral hemorrhage inflammation is the key point in cerebral hemorrhage related research, which not only can provide more theoretical basis for diagnosing the severity and prognosis of cerebral hemorrhage, but also can provide new diagnosis and treatment methods for patients with cerebral hemorrhage.
Apoptosis and necrosis are present after cerebral hemorrhage. Following apoptosis or necrosis, a number of cell signaling molecules are released which may trigger inflammation, known as risk-related molecules, including high mobility group box 1 (HMGB 1), heat shock proteins, cold shock proteins, etc., while increased HMGB1, CIRP may stimulate Toll-like receptors to induce an inflammatory response. CIRP (cold shock protein) is a stress response protein that can be activated by various cellular stressors and is acted upon by cells in response to the specific cellular environment, i.e. the specific presence of other stress response molecules, and is widely involved in a variety of physiological and pathological processes in the body. CIRP is a novel inflammation medium, can be secreted and released from heart and liver into circulation system in hemorrhagic shock and septicemia, and plays a role of a Damage-associated molecular protein (DAMP) to trigger inflammatory response by mediating the formation of a compound (TLR 4-MD 2) of myeloid differentiation protein 2 (MD 2) and Toll-like receptor 4 (TLR 4) to activate a downstream NF-KB signal pathway to up-regulate the expression of TNF-alpha and HMGB 1. CIRP has now been identified as an important mediator in severe inflammation or ischemia, with a deleterious function of exacerbating cellular injury. Meanwhile, the research proves that MD2 becomes one of the very potential targets for treating inflammatory reaction, and the targeted inhibition of MD2 can effectively inhibit the inflammatory reaction induced by MD2/TLR4/NF-KB signal channel.
A study on hemorrhagic shock by Nature Medicine has found that neutralization of CIRP secreted in hemorrhagic shock and sepsis serum by anti-CIRP antibody therapy can significantly reduce the occurrence of systemic and local inflammatory responses. They found that MD2 specifically binds to one of CIRP and has higher affinity than TLR4 and MD 2. And demonstrates that CIRP can mediate the binding of MD2 to TLR4 to trigger an inflammatory response. Thus, we suggest that we can inhibit its mediated pro-inflammatory response in inflammatory conditions by inhibiting CIRP binding to MD2 in microglia.
The mortality rate of patients with cerebral hemorrhage is high, most survivors have neurological dysfunction and bring huge material and labor cost, and the key mechanism of cerebral hemorrhage is still unclear at present; in addition to supportive treatment, there is currently a lack of an exact effective treatment. Therefore, cerebral hemorrhage is not only a clinical problem to be overcome, but also a significant public health problem.
Disclosure of Invention
The invention provides a polypeptide analogue and application thereof in a medicament for intervening and treating cerebral hemorrhage, and solves the technical problems that the key mechanism of cerebral hemorrhage in the prior art is still unclear, an exact and effective treatment method is lacked at present and the like.
The invention adopts the following technical scheme: a polypeptide analogue, wherein the polypeptide in the polypeptide analogue is Tat-CIRP-CMA, wherein Tat is a transmembrane structural sequence of 11 amino acids, cold-inducible protein CIRP is a 15 amino acid sequence on the cold-inducible protein CIRP with the highest affinity with MD2, and CMA is a signal peptide sequence of 14 amino acids targeting proteasome.
As a preferred technical scheme of the invention: the 15 amino acid sequences with the highest affinity: GRGFS RGGGD RGYGG are provided.
As a preferred technical scheme of the invention: the polypeptide analogs include: an amino acid fragment in the transcriptional transactivator Tat, a 15 amino acid fragment on the cold inducible protein CIRP with the highest affinity for MD2, and a key sequence KFERQKILDQRFFE necessary for chaperone-mediated autophagy.
As a preferred technical scheme of the invention: the 47-57 amino acid fragment of the transcriptional transactivator Tat is located at the N-terminus of the fusion protein, the 111-125 amino acid fragment of the cold inducible protein CIRP is located in the middle of the fusion protein, and the key sequence KFERQKILDQRFFE necessary for chaperone-mediated autophagy is located at the C-terminus of the fusion protein.
As a preferred technical scheme of the invention: the trans-activator of transcription Tat can enter the interior of a cell through a membrane and has strong internalization; the Tat protein consists of 86-102 amino acid residues, and a 47-57 amino acid fragment in the Tat protein is selected, wherein the 47-57 amino acid fragment is abbreviated as YGRKKRRQRRR, and is specifically Try-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.
As a preferred technical scheme of the invention: the 111-position 125 amino acid fragment in the cold-induced protein CIRP can effectively identify MD2 and combine with MD2, and the 111-position 125 amino acid fragment is abbreviated as GRGFSRGGGDRGYGG, and is specifically Gly-Arg-Gly-Phe-Ser-Arg-Gly-Gly-Asp-Arg-Gly-Tyr-Gly-Gly.
As a preferred technical scheme of the invention: the key sequence KFERQKILDQRFFE can induce molecular Chaperone Mediated Autophagy (CMA), which is the selective degradation of protein with KFERQKILDQRFFE sequence in cytoplasm through a lysosome pathway to achieve the purpose of clearance; selecting a key sequence KFERQKILDQRFFE in inducing Chaperone Mediated Autophagy (CMA), in particular Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu.
As a preferred technical scheme of the invention: an amino acid fragment in a transcription trans-activation protein Tat in the polypeptide analogue drives the polypeptide analogue to penetrate and enter cells, an amino acid fragment in a cold-induced protein CIRP can recognize MD2 and combine with MD2, a key sequence KFERQKILDQRFFE in molecular chaperone-mediated autophagy (CMA) is induced to drive the polypeptide analogue combined with MD2 to enter proteasomes for degradation, the polypeptide specifically targets MD2, specifically binds MD2, and introduces MD2 into proteasomes for degradation, so that the formation of an MD2-TLR4 complex is inhibited, a TLR 4/NF-KB-mediated inflammatory reaction is further inhibited, the function of M1 type microglia is inhibited, and secondary inflammatory injury after cerebral hemorrhage is improved; the polypeptide analogue treatment can improve the spatial learning and memory ability of mice and relieve anxiety; the therapeutic effect of the polypeptide analogue is related to inhibiting inflammatory reaction and promoting microglial cell transformation.
The application of a polypeptide analogue in the medicine for intervening and treating cerebral hemorrhage comprises the following steps:
first, it was investigated whether CIRP aggravates the inflammatory response after cerebral hemorrhage by directly modulating the conversion of microglia into "harmful" M1 type: the whole experiment constructs a rat basement membrane cerebral hemorrhage model of autoblood injection, a sham operation group, a cerebral hemorrhage group and a cerebral hemorrhage administration random polypeptide control group are adopted, the cerebral hemorrhage and administration polypeptide intervention group and MD2 or CIRP knock-out cerebral hemorrhage group are adopted for treatment, wherein the polypeptide intervention group adopts 6-hour intravenous injection after Tat-CIRP-CMA (10 mg/Kg) cerebral hemorrhage;
secondly, the influence of the behavioral detection polypeptide intervention group on the neurobehavioral prognosis of the rat is mainly shown in the improvement condition of the rat limb movement disorder: after rat blood is obtained, detecting the contents of TNF-alpha, HMGB1, CIRP and MD2 in each group of serum by an Elisa method, obtaining brain tissue, measuring the water content of the brain tissue, and detecting the hematoma volume by using an ultra-high resolution small animal ultrasonic imaging system;
the third step: separating a cortex, and measuring mRNA and protein expression conditions of CIRP, MD2, TNF-alpha and HMGB 1;
the fourth step: immunohistochemical staining examined the proportion of cells of type M1 and M2 in the cortex;
the fifth step: detecting the proportion of M1 type microglia and M2 type microglia in rat whole brain single cell suspension by a flow cytometer;
and a sixth step: the CIRP/MD2 is confirmed to participate in regulating the conversion of the microglia to the M1 type, and the polypeptide stem prognosis can effectively inhibit the generation of the M1 type microglia and reduce the brain injury;
seventh step, the effect of Tat-CIRP-CMA on the learning and memory functions of cerebral hemorrhage rats: after 21 days of cerebral hemorrhage, the water maze test result is adopted to show that in the learning and training process, the time of the contrast group reaching the platform at the appointed position is obviously prolonged, which prompts that the cerebral hemorrhage causes hippocampal damage to seriously affect the long-term learning and memory function, the treatment group animals start from the 3 rd day of self-learning training, the latency reaching the platform is obviously shortened (P <0.01 or P < 0.05) compared with the contrast group, after the learning and training of the first 5 days are finished, after the platform is removed on the 6 th day, the staying time ratio of the sham operation group in the target quadrant is 38.7% +/-3.5%, the control group decreased to 18.6% + -1.9%, while the Tat-CIRP-CMA treated group increased to 26.7% + -2.5%, with significant statistical significance compared to the control group (P < 0.05), therefore, the Tat-CIRP-CMA intervention can effectively improve the learning and memory dysfunction caused by cerebral hemorrhage;
eighth step, the expression of mRNA of the rat cortical inflammation related factor is changed at different time of cerebral hemorrhage: after rat autoblood is injected with basement membrane to build a cerebral hemorrhage model at different time, RNA is extracted from cerebral cortex, and the expression condition of inflammation-related factor mRNA is detected by Real-PCR, and the result shows that the expression of CIRP, MD2 and TNF alpha mRNA is obviously increased compared with that of a sham operation group after 3d of cerebral hemorrhage;
ninth, through interfering or reducing biological function detection of CIRP/MD2 after cerebral hemorrhage, TLR4/NF-KB signal channel is inhibited, further inflammatory mediators are inhibited from forming cascade waterfall effect, and damage of secondary injury to the central nervous system is reduced: in vitro experiments, after cerebral hemorrhage and cell inflammation models are respectively constructed on rat primary-obtained microglia by using heme (60 uM) or lipopolysaccharide (LPS, 1ug/ul, 6 h), Tat-CIRP polypeptide treatment (specificity is given to down-regulate the effect of endogenous CIRP) is carried out, and the proinflammatory effect of CIRP on the microglia and the mechanism thereof are investigated;
the tenth step: detecting the contents of TNF-alpha, HMGB1, CIRP and MD2 in cell supernatant by an Elisa method, and determining the mRNA and protein expression conditions of CIRP, MD2, TNF-alpha and HMGB1 in microglia;
the eleventh step: detecting the proportion of M1 type microglia and M2 type microglia in rat whole brain single cell suspension by a flow cytometer;
the twelfth step: through blocking a TLR4 receptor, a specific signal path of the proinflammatory effect of the CIRP is confirmed, the content of the endogenous CIRP is confirmed to intervene, so that the inflammatory reaction can be effectively inhibited, the conversion of the M1 type microglia to the M2 type is promoted, and the cerebral hemorrhage prognosis is improved;
the thirteenth step: in vitro results show that the phagocytic capacity of the microglia of M1 type is obviously reduced compared with that of M0 type (unstimulated normal microglia), while the phagocytic capacity of the microglia of M2 type is increased by 2 to 3 times compared with that of M1 type;
the fourteenth step is that: the 3-day post-treatment group of rats with Tat-CIRP-CMA cerebral ischemia had a significantly reduced production of M1 microglia in the brain compared to the control group.
Has the advantages that:
compared with the prior art, the polypeptide analogue and the application thereof in the medicine for intervening and treating cerebral hemorrhage have the following technical effects:
1. the polypeptide analogue has good treatment effect on a cerebral hemorrhage model;
2. at present, no research report for treating cerebral hemorrhage inflammatory reaction by using a polypeptide analogue exists, and the researched polypeptide analogue can further play an effective pharmaceutical role;
3. the invention provides a strategy for clinical treatment of cerebral hemorrhage, and has very important practical value;
4. the treatment cost of cerebral hemorrhage is high, the medical resource consumption is large, the life quality of human beings is seriously influenced, and great threat is already caused to the human health;
5. the Tat-CIRP-CMA is intravenously administered for 2 hours after cerebral hemorrhage, so that learning and memory dysfunction caused by cerebral hemorrhage can be effectively improved, and anxiety can be relieved.
6. In vitro results demonstrated that after 3 days of treatment of rats with cerebral ischemia with Tat-CIRP-CMA, the production of M1 microglia in the brain was significantly reduced compared to the control group.
7. The rats with cerebral ischemia treated by Tat-CIRP-CMA can effectively improve secondary inflammatory injury after cerebral hemorrhage.
Drawings
FIG. 1 is a graph showing the effect of Tat-CIRP-CMA on learning and memory function in rats with cerebral hemorrhage.
FIG. 2 is a graph showing the expression change of mRNA, a factor associated with inflammation of rat cortex, at different times of cerebral hemorrhage in the present application.
FIG. 3 is a graph showing the conversion of rat microglia to type M1 after Tat-CIRP-CMA of the present application inhibits cerebral hemorrhage.
Detailed Description
The present invention is further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the scope of the claims, and other alternatives which may occur to those skilled in the art are within the scope of the claims.
Example 1: a polypeptide analogue, wherein the polypeptide in the polypeptide analogue is Tat-CIRP-CMA, wherein Tat is a transmembrane structural sequence of 11 amino acids, CIRP is a 15 amino acid sequence on cold-inducible protein CIRP with highest affinity with MD2, and CMA is a signal peptide sequence of 14 amino acids targeting proteasome.
The 15 amino acid sequences with the highest affinity: GRGFS RGGGD RGYGG are provided.
The polypeptide analogs include: an amino acid fragment in the transcriptional transactivator Tat, an amino acid fragment in the cold-inducible protein CIRP, and a key sequence KFERQKILDQRFFE necessary for chaperone-mediated autophagy.
The 47-57 amino acid fragment of the transcriptional transactivator Tat is located at the N-terminus of the fusion protein, the 111-125 amino acid fragment of the cold inducible protein CIRP is located in the middle of the fusion protein, and the key sequence KFERQKILDQRFFE necessary for chaperone-mediated autophagy is located at the C-terminus of the fusion protein.
The trans-activator of transcription Tat can enter the interior of a cell through a membrane and has strong internalization; the Tat protein consists of 86-102 amino acid residues, and a 47-57 amino acid fragment in the Tat protein is selected, wherein the 47-57 amino acid fragment is abbreviated as YGRKKRRQRRR, and is specifically Try-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.
The 111-position 125 amino acid fragment in the cold-induced protein CIRP can effectively identify MD2 and combine with MD2, and the 111-position 125 amino acid fragment is abbreviated as GRGFSRGGGDRGYGG, and is specifically Gly-Arg-Gly-Phe-Ser-Arg-Gly-Gly-Asp-Arg-Gly-Tyr-Gly-Gly.
The key sequence KFERQKILDQRFFE can induce molecular Chaperone Mediated Autophagy (CMA), which is the selective degradation of protein with KFERQKILDQRFFE sequence in cytoplasm through a lysosome pathway to achieve the purpose of clearance; selecting a key sequence KFERQKILDQRFFE in inducing Chaperone Mediated Autophagy (CMA), in particular Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu.
An amino acid fragment in a transcription trans-activation protein Tat in the polypeptide analogue drives the polypeptide analogue to penetrate and enter cells, an amino acid fragment in a cold-induced protein CIRP can recognize MD2 and combine with MD2, a key sequence KFERQKILDQRFFE in molecular chaperone-mediated autophagy (CMA) is induced to drive the polypeptide analogue combined with MD2 to enter proteasomes for degradation, the polypeptide specifically targets MD2, specifically binds MD2, and introduces MD2 into proteasomes for degradation, so that the formation of an MD2-TLR4 complex is inhibited, a TLR 4/NF-KB-mediated inflammatory reaction is further inhibited, the function of M1 type microglia is inhibited, and secondary inflammatory injury after cerebral hemorrhage is improved; the polypeptide analogue treatment can improve the spatial learning and memory ability of mice and relieve anxiety; the therapeutic effect of the polypeptide analogue is related to inhibiting inflammatory reaction and promoting microglial cell transformation.
The application of the polypeptide analogue in the medicine for intervening and treating cerebral hemorrhage comprises the following steps:
first, it was investigated whether CIRP aggravates the inflammatory response after cerebral hemorrhage by directly modulating the conversion of microglia into "harmful" M1 type: the whole experiment constructs a rat basement membrane cerebral hemorrhage model of autoblood injection, a sham operation group, a cerebral hemorrhage group and a cerebral hemorrhage administration random polypeptide control group are adopted, the cerebral hemorrhage and administration polypeptide intervention group and MD2 or CIRP knock-out cerebral hemorrhage group are adopted for treatment, wherein the polypeptide intervention group adopts Tat-CIRP-CMA (10 mg/Kg) cerebral hemorrhage followed by intravenous injection for 2 hours;
secondly, the influence of the behavioral detection polypeptide intervention group on the neurobehavioral prognosis of the rat is mainly shown in the improvement condition of the rat limb movement disorder: after rat blood is obtained, detecting the contents of TNF-alpha, HMGB1, CIRP and MD2 in each group of serum by an Elisa method, obtaining brain tissue, measuring the water content of the brain tissue, and detecting the hematoma volume by using an ultra-high resolution small animal ultrasonic imaging system;
the third step: separating a cortex, and measuring mRNA and protein expression conditions of CIRP, MD2, TNF-alpha and HMGB 1;
the fourth step: immunohistochemical staining examined the proportion of cells of type M1 and M2 in the cortex;
the fifth step: detecting the proportion of M1 type microglia and M2 type microglia in rat whole brain single cell suspension by a flow cytometer;
and a sixth step: the CIRP/MD2 is confirmed to participate in regulating the conversion of the microglia to the M1 type, and the polypeptide stem prognosis can effectively inhibit the generation of the M1 type microglia and reduce the brain injury;
seventh step, the effect of Tat-CIRP-CMA on the learning and memory functions of cerebral hemorrhage rats: as shown in fig. 1, 21 days after cerebral hemorrhage, the water maze test result is adopted to show that in the process of learning and training, the time of the contrast group reaching the platform at the designated position is obviously prolonged, which indicates that the hippocampus damage caused by cerebral hemorrhage seriously influences the long-term learning and memory functions, the incubation period of the animals in the treatment group reaching the platform is obviously shortened (P is less than 0.01 or P is less than 0.05) compared with that in the contrast group from the 3 rd day of the self-learning training of the animals in the treatment group, after the learning and training of the first 5 days are finished, after the platform is removed on the 6 th day, the staying time ratio of the sham operation group in the target quadrant is 38.7% +/-3.5%, the control group decreased to 18.6% + -1.9%, while the Tat-CIRP-CMA treated group increased to 26.7% + -2.5%, with significant statistical significance compared to the control group (P < 0.05), therefore, the Tat-CIRP-CMA intervention can effectively improve the learning and memory dysfunction caused by cerebral hemorrhage;
eighth step, the expression of mRNA of the rat cortical inflammation related factor is changed at different time of cerebral hemorrhage: as shown in fig. 2, after rat autoblood is injected with basement membrane to build a cerebral hemorrhage model at different times, RNA is extracted from cerebral cortex, and Real-PCR is performed to detect the expression of inflammation-related factor mRNA, and the results show that CIRP, MD2, TNF α mRNA expression is significantly up-regulated compared with that of a sham operation group after 3d cerebral hemorrhage;
ninth, through interfering or reducing biological function detection of CIRP/MD2 after cerebral hemorrhage, TLR4/NF-KB signal channel is inhibited, further inflammatory mediators are inhibited from forming cascade waterfall effect, and damage of secondary injury to the central nervous system is reduced: in vitro experiments, after cerebral hemorrhage and cell inflammation models are respectively constructed on rat primary-obtained microglia by using heme (60 uM) or lipopolysaccharide (LPS, 1ug/ul, 6 h), Tat-CIRP polypeptide treatment (specificity is given to down-regulate the effect of endogenous CIRP) is carried out, and the proinflammatory effect of CIRP on the microglia and the mechanism thereof are investigated;
the tenth step: detecting the contents of TNF-alpha, HMGB1, CIRP and MD2 in cell supernatant by an Elisa method, and determining the mRNA and protein expression conditions of CIRP, MD2, TNF-alpha and HMGB1 in microglia;
the eleventh step: detecting the proportion of M1 type microglia and M2 type microglia in rat whole brain single cell suspension by a flow cytometer;
the twelfth step: through blocking a TLR4 receptor, a specific signal path of the proinflammatory effect of the CIRP is confirmed, the content of the endogenous CIRP is confirmed to intervene, so that the inflammatory reaction can be effectively inhibited, the conversion of the M1 type microglia to the M2 type is promoted, and the prognosis of cerebral hemorrhage is improved;
the thirteenth step: as shown in fig. 3A, in vitro results confirmed that M1-type microglia significantly decreased phagocytic ability compared to M0-type (unstimulated normal microglia), while M2-type increased phagocytic ability 2-to 3-fold compared to M1-type;
the fourteenth step is that: as shown in FIG. 3B, after 3 days of treatment of rats with cerebral ischemia by Tat-CIRP-CMA, there was a significant reduction in the production of M1 microglia in the brain as compared to the control group.
The cerebral hemorrhage treatment cost is high, the medical resource consumption is large, the life quality of human beings is seriously influenced, and great threat is already caused to the human health. The invention provides a strategy for clinical treatment of cerebral hemorrhage, and has important practical value.
Sequence listing
<110> university of southeast Tong
<120> polypeptide analogue and application thereof in medicine for intervening and treating cerebral hemorrhage
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Claims (9)
1. A polypeptide analog, characterized by: the polypeptide in the polypeptide analogue is Tat-CIRP-CMA, wherein Tat is a transmembrane structure sequence of 11 amino acids, cold-induced protein CIRP is a 15 amino acid sequence with highest affinity with MD2, and CMA is a signal peptide sequence of 14 amino acids of a targeted proteasome.
2. The polypeptide analog of claim 1, wherein: the 15 amino acid sequences with the highest affinity: GRGFS RGGGD RGYGG are provided.
3. The polypeptide analog of claim 1, wherein: the polypeptide analogs include: an amino acid fragment in the transcriptional transactivator Tat, a 15 amino acid sequence on the cold inducible protein CIRP with the highest affinity to MD2, and a key sequence KFERQKILDQRFFE necessary for chaperone-mediated autophagy.
4. The polypeptide analog of claim 3, wherein: the 47-57 amino acid fragment of the transcriptional transactivator Tat is located at the N-terminus of the fusion protein, the 111-125 amino acid fragment of the cold inducible protein CIRP is located in the middle of the fusion protein, and the key sequence KFERQKILDQRFFE necessary for chaperone-mediated autophagy is located at the C-terminus of the fusion protein.
5. The polypeptide analog of claim 4, wherein: the trans-activator of transcription Tat can enter the interior of a cell through a membrane and has strong internalization; the Tat protein consists of 86-102 amino acid residues, and a 47-57 amino acid fragment in the Tat protein is selected, wherein the 47-57 amino acid fragment is abbreviated as YGRKKRRQRRR, and is specifically Try-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.
6. The polypeptide analog of claim 4, wherein: the 111-position 125 amino acid fragment of the cold-inducible protein CIRP can effectively identify MD2 and combine with MD2, and the 111-position 125 amino acid fragment is abbreviated as GRGFSRGGGDRGYGG, and is specifically Gly-Arg-Gly-Phe-Ser-Arg-Gly-Gly-Asp-Arg-Gly-Tyr-Gly-Gly.
7. The polypeptide analog of claim 4, wherein: the key sequence KFERQKILDQRFFE can induce molecular Chaperone Mediated Autophagy (CMA), which is the selective degradation of protein with KFERQKILDQRFFE sequence in cytoplasm through a lysosome pathway to achieve the purpose of clearance; selecting a key sequence KFERQKILDQRFFE in inducing Chaperone Mediated Autophagy (CMA), in particular Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu.
8. The polypeptide analog of claim 4, wherein: an amino acid fragment in a transcription trans-activation protein Tat in the polypeptide analogue drives the polypeptide analogue to penetrate and enter cells, an amino acid fragment in a cold-induced protein CIRP can recognize MD2 and combine with MD2, a key sequence KFERQKILDQRFFE in molecular chaperone-mediated autophagy (CMA) is induced to drive the polypeptide analogue combined with MD2 to enter proteasomes for degradation, the polypeptide specifically targets MD2, specifically binds MD2, and introduces MD2 into proteasomes for degradation, so that the formation of an MD2-TLR4 complex is inhibited, a TLR 4/NF-KB-mediated inflammatory reaction is further inhibited, the function of M1 type microglia is inhibited, and secondary inflammatory injury after cerebral hemorrhage is improved; the polypeptide analogue treatment can improve the spatial learning and memory ability of mice and relieve anxiety; the therapeutic effect of the polypeptide analogue is related to inhibiting inflammatory reaction and promoting microglial cell transformation.
9. The use of the polypeptide analog of claim 4 in a medicament for the interventional treatment of cerebral hemorrhage, comprising the steps of:
first, it was investigated whether CIRP aggravates the inflammatory response after cerebral hemorrhage by directly modulating the conversion of microglia into "harmful" M1 type: the whole experiment constructs a rat basement membrane cerebral hemorrhage model of autoblood injection, a sham operation group, a cerebral hemorrhage group and a cerebral hemorrhage administration random polypeptide control group are adopted, the cerebral hemorrhage and administration polypeptide intervention group and MD2 or CIRP knock-out cerebral hemorrhage group are adopted for treatment, wherein the polypeptide intervention group adopts Tat-CIRP-CMA (10 mg/Kg) cerebral hemorrhage followed by intravenous injection for 2 hours;
secondly, the influence of the behavioral detection polypeptide intervention group on the neurobehavioral prognosis of the rat is mainly shown in the improvement condition of the rat limb movement disorder: after rat blood is obtained, detecting the contents of TNF-alpha, HMGB1, CIRP and MD2 in each group of serum by an Elisa method, obtaining brain tissue, measuring the water content of the brain tissue, and detecting the hematoma volume by using an ultra-high resolution small animal ultrasonic imaging system;
the third step: separating a cortex, and measuring mRNA and protein expression conditions of CIRP, MD2, TNF-alpha and HMGB 1;
the fourth step: immunohistochemical staining examined the proportion of cells of type M1 and M2 in the cortex;
the fifth step: detecting the proportion of M1 type microglia and M2 type microglia in rat whole brain single cell suspension by a flow cytometer;
and a sixth step: the CIRP/MD2 is confirmed to participate in regulating the conversion of the microglia to the M1 type, and the polypeptide stem prognosis can effectively inhibit the generation of the M1 type microglia and reduce the brain injury;
seventh step, the effect of Tat-CIRP-CMA on the learning and memory functions of cerebral hemorrhage rats: after 21 days of cerebral hemorrhage, the water maze test result is adopted to show that in the learning and training process, the time of the contrast group reaching the platform at the appointed position is obviously prolonged, which prompts that the cerebral hemorrhage causes hippocampal damage to seriously affect the long-term learning and memory function, the treatment group animals start from the 3 rd day of self-learning training, the latency reaching the platform is obviously shortened (P <0.01 or P < 0.05) compared with the contrast group, after the learning and training of the first 5 days are finished, after the platform is removed on the 6 th day, the staying time ratio of the sham operation group in the target quadrant is 38.7% +/-3.5%, the control group decreased to 18.6% + -1.9%, while the Tat-CIRP-CMA treated group increased to 26.7% + -2.5%, with significant statistical significance compared to the control group (P < 0.05), therefore, the Tat-CIRP-CMA intervention can effectively improve the learning and memory dysfunction caused by cerebral hemorrhage;
eighth step, the expression of mRNA of the rat cortical inflammation related factor is changed at different time of cerebral hemorrhage: after rat autoblood is injected with basement membrane to build a cerebral hemorrhage model at different time, RNA is extracted from cerebral cortex, and the expression condition of inflammation-related factor mRNA is detected by Real-PCR, and the result shows that the expression of CIRP, MD2 and TNF alpha mRNA is obviously increased compared with that of a sham operation group after 3d of cerebral hemorrhage;
ninth, through interfering or reducing biological function detection of CIRP/MD2 after cerebral hemorrhage, TLR4/NF-KB signal channel is inhibited, further inflammatory mediators are inhibited from forming cascade waterfall effect, and damage of secondary injury to the central nervous system is reduced: in vitro experiments, after cerebral hemorrhage and cell inflammation models are respectively constructed on rat primary-obtained microglia by using heme (60 uM) or lipopolysaccharide (LPS, 1ug/ul, 6 h), Tat-CIRP polypeptide treatment (specificity is given to down-regulate the effect of endogenous CIRP) is carried out, and the proinflammatory effect of CIRP on the microglia and the mechanism thereof are investigated;
the tenth step: detecting the contents of TNF-alpha, HMGB1, CIRP and MD2 in cell supernatant by an Elisa method, and determining the mRNA and protein expression conditions of CIRP, MD2, TNF-alpha and HMGB1 in microglia;
the eleventh step: detecting the proportion of M1 type microglia and M2 type microglia in rat whole brain single cell suspension by a flow cytometer;
the twelfth step: through blocking a TLR4 receptor, a specific signal path of the proinflammatory effect of the CIRP is confirmed, the content of the endogenous CIRP is confirmed to intervene, so that the inflammatory reaction can be effectively inhibited, the conversion of the M1 type microglia to the M2 type is promoted, and the cerebral hemorrhage prognosis is improved;
the thirteenth step: in vitro results show that the phagocytic capacity of the M1 type microglia is obviously reduced compared with that of the M0 type, namely unstimulated normal microglia, while the phagocytic capacity of the M2 type microglia is increased by 2 to 3 times compared with that of the M1 type microglia;
the fourteenth step is that: after 3 days of cerebral ischemia of rats, the observation shows that after the treatment by using Tat-CIRP-CMA, compared with a control, the generation of M1 type microglia in the brain can be obviously reduced.
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