CN111166885B - Neuroprotective agents and uses thereof - Google Patents

Abstract

The invention discloses a neuroprotective agent, which is selected from the following group: drugs for inhibiting dissociation of the N-terminal and the C-terminal of the subunit ASIC1a under the acidosis condition; or a drug that prevents the dissociated C-terminal toxic fragment of ASIC1a from binding to RIPK 1; or drugs which prevent the dissociated ASIC1a C terminal toxic fragment from being combined with RIPK1 and inhibit the dissociation of ASIC1a subunit N terminal and C terminal under the acidosis condition. Pharmaceutical compositions comprising the neuroprotective agents are also disclosed. The nerve protective agent of the invention has protective effect on neurons in acid toxicity and cerebral ischemia injury, is expected to become a medicament for treating and preventing clinical stroke diseases, and has very wide application prospect.

Description

Neuroprotective agents and uses thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a neuroprotective agent and application thereof.

Background

Apoplexy is one of the diseases with the highest global fatality rate and disability rate, and the life risk and the treatment of sequelae in the recovery period brought by acute apoplexy bring heavy economic burden to the society. China is the country with the highest incidence of stroke, mortality and disability rate all over the world. Today, stroke has become the most deaths annually in china and is a major challenge facing "healthy china".

Clinically, 80 percent of the patients are ischemic stroke, and the treatment measures for acute ischemic stroke mainly comprise two measures, one is to use tissue plasminogen activator (t-PA) to carry out thrombolysis, and the other is to take out the thrombus by machinery and then carry out recanalization. Thrombolytic therapy is only 5% of patients who can perform this method due to the time window limitation, and is prone to triggering ischemia-reperfusion injury and hemorrhagic transformation. Cerebral ischemia triggers a complex pathological biochemical cascade. Excitotoxicity, acidosis, oxidative stress, inflammatory reaction and the like mediate different neuronal death modes such as apoptosis, necrosis, autophagy and the like. Therefore, the neuroprotective treatment for the neuron death has wide application prospect. However, N-methyl-D-aspartate receptor (NMDAR), which is involved in neuronal excitotoxicity in ischemic stroke, has failed in clinical trials for stroke, mainly because classical ion channels such as NMDA receptor perform very important physiological functions and must cause serious side effects once damaged, and thus research on new mechanisms of neuronal death after stroke and development of neuroprotective agents targeting the same are urgently needed.

During cerebral ischemia, the cerebral tissue can not carry out normal aerobic metabolism, and the pH value (pH value) of the ischemic tissue and the periphery is remarkably reduced to about 6.5-6.0, so that the acidosis is considered to be one of the main mechanisms for causing ischemic brain injury. Among the numerous mechanisms of proton sensing, acid-toxic damage mediated by acid-sensitive ion channels (ASICs), particularly their subunit acid-sensitive ion channel 1a (ASIC1a), is a major cause of neuronal death. When the extracellular pH is lowered, ASIC1a is activated and an inward current consisting primarily of sodium ions and a small portion of calcium ions is generated. Traditionally, this calcium influx was thought to cause cytosolic calcium overload leading to cell death, and work published in this laboratory 2015 suggested that ASIC1 a-mediated neuronal acid toxic death, which could be inhibited by the inhibitor Nec-1 of key protein receptor interacting protein kinase 1(RIPK1) in the programmed necrosis pathway, could be independent of the involvement of ionic signals, and that allosteric signaling of ASIC1a played a role in the death pathway. CP-1 polypeptides that mimic the C-terminal amino acid fragment of ASIC1a are capable of mimicking acid-toxin mediated death. In the middle wind H⁺The increase not only activates the ion signal pathway of ASIC1a, but also changes the protein conformation, so that the original embedded CP-1 fragment at the C end of ASIC1a is exposed, and is specifically combined with RIPK1, and the latter is phosphorylated, thereby activating the programmed necrosis process of neurons to cause injury.

Although this is not a general phenomenon, studies have shown that channel-independent conformational signaling plays an important role in the functional execution of other classical ion channels, such as voltage-dependent calcium channels, NMDA receptors, and KA receptor channels. The ion channel allosteric signal has universality. These reports highlight the diversity of membrane protein signals: even if the same stimulus is received, the same protein performs either a channel function or a non-channel function, and the downstream can produce distinct responses. However, the mechanism by which the channel performs its non-ionic channel function is unclear.

Disclosure of Invention

The invention aims to solve the technical problem of lack of effective medicines for treating cerebral apoplexy diseases at present, and provides a nerve protective agent which has a protective effect on neurons in acidosis and cerebral ischemia injury and is expected to become a medicine for treating and preventing clinical ischemic apoplexy diseases.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the present invention, there is provided a neuroprotective agent selected from the group consisting of:

(1) drugs for inhibiting dissociation of the N-terminal and the C-terminal of the subunit ASIC1a under the acidosis condition;

(2) a drug that prevents the dissociated ASIC1a C-terminal toxic fragment from binding to RIPK 1;

(3) drugs that prevent the dissociated C-terminal toxic fragment of ASIC1a from binding to RIPK1 and inhibit the dissociation of the N-terminal and C-terminal of ASIC1a subunit under acidogenic conditions.

Preferably, the neuroprotective agent comprises an NSF (N-ethylmaleimide sensitive fusion protein) inhibitor, an NSF knockdown shRNA, a transmembrane protective peptide mimicking the N-terminus of ASIC1 a.

Preferably, the transmembrane protection peptide mimicking the N-terminus of ASIC1a comprises at least four glutamates at positions 6 to 9 of the N-terminus of ASIC1 a. The transmembrane protective peptide comprises a TAT membrane-entering sequence.

More preferably, the transmembrane protection peptide has an amino acid sequence shown in SEQ ID NO. 1.

Preferably, the NSF inhibitor is N-ethylmaleimide (NEM).

In the present invention, the term "acid-sensing ion channels" (ASICs) refers to a class of cation-permeable protein complexes widely present on cell membranes, belonging to the epithelial sodium channel/degenerated protein superfamily, and having important roles in sensing the pH of body fluid and regulating multiple physiological functions such as pain sensation and mechanical sensation.

Molecular cloning has shown that ASIC has at least six subunits encoded by four genes (ASIC1a,1b,2a,2b,3 and 4) that form homotrimeric or heterotrimeric functional complexes. The diversity of ASIC subunit expression and distribution, as well as the variation of subunit composition and channel gating, allows for diversity in channel function.

ASIC1a has a distribution in both the central and peripheral nervous systems, primarily involved in synaptic transmission and plasticity. ASIC1a can be used for treating various neurological diseases such as ischemic cell death, epilepsy, and neurodegenerative diseases. The ASIC1a channel referred to herein refers to a homopolymer or a heteromer composed primarily of ASIC1a subunits.

In another aspect of the present invention, there is also provided a pharmaceutical composition for treating and/or preventing cerebral stroke diseases, comprising a safe and effective amount of the above neuroprotective agent, and a pharmaceutically acceptable carrier.

Such acceptable carriers are non-toxic, can be adjunctive to administration, and do not adversely affect the therapeutic efficacy of the neuroprotective agents of the present invention. Such carriers can be any solid excipient, liquid excipient, semi-solid excipient, or in aerosol compositions, gaseous excipient, commonly available to those skilled in the art.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional methods in the pharmaceutical field. For example, the neuroprotective agent can be admixed with one or more carriers and then formulated into a desired dosage form, such as tablets, pills, capsules, semisolids, powders, sustained release dosage forms, solutions, suspensions, formulations, aerosols, and the like.

In another aspect of the present invention, there is also provided the use of the above neuroprotective agent in the manufacture of a medicament for the treatment and/or prevention of stroke disorders.

In another aspect of the invention, there is also provided the use of a neuroprotective agent as described above in combination with at least one thrombolytic agent in the preparation of a medicament for the treatment of stroke disorders.

The neuroprotective agent provided by the invention has a protective effect on neurons in acid toxicity and cerebral ischemic injury by inhibiting the dissociation of the N end and the C end of the ASIC1a under the acid toxicity condition or preventing the dissociated toxic fragment of the C end of the ASIC1a from being combined with the RIPK1, provides a new medicine or target for treating or preventing clinical ischemic stroke diseases, and has a very wide application prospect.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 shows a polypeptide NT of example 1 of the present invention_1-20An experimental result diagram with protective effect on the acid-toxin-mediated programmed necrosis of mouse cortical neurons;

FIG. 2 is a graph of the experimental results of the critical importance of ASIC1a N remote sequence to inhibit CP-1 death fragment in example 2 of the present invention;

FIG. 3 is a graph showing the results of experiments conducted by ischemic lateral cerebellar ASIC1a in accordance with example 3 of the present invention in recruiting NSF to form the NSF-ASIC1a-RIPK1 complex;

FIG. 4 is a graph of the results of an experiment in which NSF of embodiment 4 of the present invention was combined with the distal end of ASIC1a N;

FIG. 5 is a graph of the results of the NSF knockdown assay of example 5 of the present invention with protective effects in a cultured neuronal acidosis model;

FIG. 6 is a graph showing the results of experiments in which glutamic acids No. 6 to 9 at the distal end of ASIC1a N according to example 6 of the present invention are critical to the formation of intramolecular self-inhibition by ASIC1 a;

FIG. 7 is a schematic diagram of the mechanism of the conformational signaling of ASIC1a under acid-toxic conditions in accordance with the present invention.

Detailed Description

The invention researches the function of ASIC1a by adopting death detection (lactate dehydrogenase release experiment, CellTiter-Blue method, PI staining, TTC staining), fluorescence energy resonance transfer (FRET), mass spectrometry, traditional biochemical means (immunoblot experiment, protein co-immunoprecipitation), Rosetta modeling, electrophysiology and other means, and researches the reason why the CP-1 toxic fragment can be in a non-activated state under physiological conditions, and how the toxic peptide is chelated by protein chaperones or amino acid fragments. The molecular mechanism underlying the conformational change of ASIC1a in case of acidification was read and the corresponding neuroprotective peptides were designed against the above findings.

Conventionally, in order to obtain stable crystals when analyzing the structure of ASIC1a protein, the intracellular N-terminus and C-terminus lacking in stability were cleaved, and the protein was purified to analyze the crystal structure. The invention fills the blank of the N terminal and C terminal structure of ASIC1a protein from the functional point of view, verifies the interaction between the N terminal and the C terminal of ASIC1a under the physiological condition, provides the regulation and control mechanism of intramolecular self-inhibition, and has important theoretical significance for the intracellular function cognition of ASIC1 a. Furthermore, the present invention confirms the important role of the conformational signaling pathway of ASIC1a in its mediation of ischemic neuronal death and defines the important role played by the involvement of NSF proteins therein. More importantly, the invention mimics the N-terminal transmembrane protective peptide NT of ASIC1a_1-20Functionally validated in vitro and in vivo models, NT_1-20Has protective effect in models of acid toxicity and cerebral ischemia injury, provides new targets and strategies for clinical treatment of ischemic stroke, and has transformation application prospect.

EXAMPLE 1 polypeptide NT_1-20Has protective effect on acid poison mediated programmed necrosis of mouse cortical neuron

The original CP-1 toxic amino acid fragment was reduced in size, and the shortest amino acid sequence CP-1-2(TATKCQKEAKRN, SEQ ID NO.2) whose C-terminal could cause cell death was found. While the presence of this amino acid sequence, ASIC1a did not lead to cell death under physiological conditions, suggesting that the C-terminal toxic fragment is covered in the full-length sequence. While terminals N and C of ASIC1a are both located intracellularly. Thus, several polypeptides were synthesized with different fragments mimicking the N-terminus of TAT membrane-entering sequence, and a polypeptide NT mimicking the N-terminal 1-20 amino acid fragment was found_1-20(TATMELKTEEEEVGGVQPVSIQA, SEQ ID NO.1) has protective effects in both CP-1-2 and in the acidosis-mediated neuronal death model (FIG. 1).

In FIG. 1, (a) corresponds to (b, c) experimental cell deathFlow chart of death analysis. (b) The CellTiter-Blue method measures the effect of peptide prediction on neuronal cell survival (concentrations were 10. mu.M each). Pre-administration of CP-1, CP-1-2, and CP-1-3 peptides results in decreased cell viability. Nec-1 (20. mu.M) reduced CP-1-2 induced cell death. CP-1-3 caused less cell death than CP-1-2, and CP-1-2S lacking the membrane-entering sequence did not cause decreased cell viability. (c) Lactate dehydrogenase release assay the protection of the N-terminal polypeptide in CP-1-2 (10. mu.M) mediated cell death model was evaluated. Toxic Effect of the polypeptide CP-1-2 by NT_1-20Can not be NT_11-30And NT_21-41And (4) inhibiting. (d) Corresponding to (e, f) experimental cell death analysis flowsheet. (e) 10 μ M NT compared to treatment with pH 7.4_1-20The pretreatment can reduce the release amount of the neuron lactate dehydrogenase under the condition of pH6.0 treatment. (f) NT_1-20The concentration dependence of the polypeptide on the inhibition of acid toxicity was 10. mu.M in all subsequent experiments. (g) Control peptide and NT_1-20Representative graph of brightfield and PI staining after polypeptide pH 7.4 and pH6.0 treatment, NT_1-20Can obviously reduce the number of neuron PI positive cells after pH6.0 treatment. (h) (g) graph statistics. PI staining NT_1-20The polypeptide has protective effect on acidification mediated neuron death. (i)10 μ M NT_1-20Polypeptide pretreatment (0.5 hr) did not affect the current induced by pH6.0 in cultured cortical neurons.

Example 2 ASIC1a N distal sequence is crucial for the inhibition of CP-1 death fragment

In view of the results of the death assays described in example 1 above, in silico predictions were made showing that the distal end of ASIC1a N binds to the C-terminus in the closed state and the two are separated in the open state, suggesting that gating-related conformational changes result in dissociation at both ends N C. Meanwhile, the prediction of the interaction between the N end and the C end of the ASIC1a is directly verified by a fluorescence energy resonance transfer method. And by externally turning the plasmid with deletion of 1-20 amino acids at the N terminal on CHO cells, the ASIC1a with deletion of the N terminal is also in a state of conformational functional activation under the condition of no proton activation, directly mediates the death of the cells, and reflects the interaction of the distal end of the ASIC1a N and the C terminal from the functional side (figure 2).

In fig. 2, (a) Rosetta modeling of full-length ASIC1 a. Grey represents closed states and blue represents open states. (b) Construction of FRET tool plasmid (CFP-ASIC1 a-YFP). Cyan fluorescent protein was attached to the N-terminus of ASIC1a, and yellow fluorescent protein was attached to the C-terminus of ASIC1 a. (c) The fluorescence intensity ratio of YFP (excitation wavelength 405nm, emission wavelength 525nm)/CFP (excitation wavelength 405nm, emission wavelength 482nm) is taken as an index for measuring fluorescence energy resonance transfer. In three groups of CHO cells: transfecting a FRET plasmid; transfecting FRET plasmid, and pre-feeding PcTX1 before experiment; transfection of the FRET-E235C/Y389C mutant plasmid (which did not undergo a conformational change upon acidification at pH 6.0) gave a pH6.0 acidification stimulus, and the fluorescence intensity of YFP recorded by the 405nm excited FRET plasmid gradually decreased, indicating that acidification gradually increased the distance between the two fragments. The dissociation of N-terminal C-terminal of E235C/Y389C mutant with inhibited conformational change and ASIC1a inhibitor PcTx1 are obviously different from that of single-turn FRET plasmid. (d) FIG. C shows a summary of the change rates of fluorescence intensity ratios after 5min of pH6.0 treatment. (e, f) transfecting wild type ASIC1a plasmid, ASIC1a N end 1-20 amino acid deletion plasmid, ASIC1a N end 1-20 amino acid deletion HIF channel function deletion mutant plasmid and ASIC1a N end 1-20 amino acid deletion plasmid, wherein the staining of Nec-1 group PI represents images (e) and summarization (f) before experiment. The ASIC1a plasmid with deletion of 1-20 amino acids at the N-terminal can directly mediate cell death without proton activation, and the death is independent of the channel function of ASIC1a (the HIF mutant plasmid is still dead) and can be inhibited by a programmed necrosis pathway inhibitor Nec-1. (g) ASIC1a N-terminal 1-20 amino acid deleted plasmid (red trace) showed a greatly reduced pH6.0 induced acid current compared to wild type ASIC1a plasmid (black trace). (h) The Western test of the plasmid with 1-20 amino acid deletions at the N end of the transfection ASIC1a shows that the expression level of the ASIC1a is obviously reduced, but the combination of the ASIC1a and the RIPK1 is obviously increased under the condition of pH 7.4. (i) (h) statistics of the amount of RIPK1 protein bound to ASIC1a in the graph.

The above results indicate that the N-terminus of ASIC1a sequesters C-terminal toxic fragments, placing the conformational function of ASIC1a in an inhibitory state, i.e., intramolecular self-inhibition. The ASIC1a undergoes allosteric activation by proton activation, causing procedural necrosis.

Example 3 ischemic lateral cerebellar ASIC1a recruitment of NSF to form NSF-ASIC1a-RIPK1 Complex

To explore the molecular mechanism of dissociation of ASIC1a N-and C-termini upon acidification, the key role played by N-ethylmaleimide sensitive fusion protein (NSF) in the middle cerebral artery embolization (MCAO) mice was clarified by comparing the differences between the ischemic and control sides and ASIC1a binding proteins using ASIC1a antibody co-immunoprecipitation coupled coomassie blue staining electrophoresis (fig. 3).

In fig. 3, (a) co-immunoprecipitation using ASIC1a antibody coupled coomassie blue staining electrophoresis compares the difference between the ischemic side and the control side of arterial ischemia model in the rat brain and ASIC1a binding protein. Running the gel revealed a significantly increased band in the ischemic cerebellum between 70-100kD molecular weight, as shown in the red box. (b) MALDI-TOF-MS analysis is carried out on the protein-containing gel with the molecular weight of about 85kD, and 18 peptide fragments in 39 peptide fragments are matched with NSF protein. (c) The co-immunoprecipitation results confirmed that: in the mouse middle cerebral artery ischemia model, the phosphorylation level of RIPK1 in the ischemic lateral half brain is increased, and the combination of ASIC1a and NSF is obviously increased. There was no significant change in total NSF protein on the ischemic side compared to the control side. (d) And (c) summarizing the data. (e) ASIC1a knockout mice had no significant increase in binding to NSF on the ischemic side compared to control side RIPK 1. (f) (e) summarizing the data. (g) The combination of NSF and RIPK1 in the PcTX1 pre-dosed acid-treated group was significantly reduced compared to the single pH6.0 acid-treated group. (h) (g) summarizing the data.

Example 4 NSF integration with ASIC1a N remote

In order to further explore the molecular mechanism of dissociation of the N-terminal and the C-terminal of ASIC1a during acidification, the 1 st to 20 th amino acid sites of the N-terminal of the binding domain of ASIC1a and NSF (N-ethylmaleimide sensitive fusion protein) were determined by GST fusion protein sedimentation technology and co-immunoprecipitation (FIG. 4).

In FIG. 4, (a) GST fusion protein sedimentation technique shows that NSF binds to GST protein linked to the N-terminus of ASIC1 a. (b) Schematic diagram of deletion mutants of different amino acid sequences at the N-terminal of ASIC1a plasmid. (c) Co-immunoprecipitation results showed that amino acid deletions from positions 1-41 and 1-20 from the N-terminus reduced acidification-mediated binding of ASIC1a to NSF. (d) And (c) summarizing the data. (e) The co-immunoprecipitation result shows that 10. mu.M NT_1-20Polypeptide preparationTreatment for 0.5 hours reduced acidification-mediated formation of the NSF-ASIC1a-RIPK1 complex and reduced acidification-mediated degradation of RIPK1 protein. (f) (e) summarizing the data.

Example 5 NSF knockdown has protective effects in cultured neuronal acidosis models

Intervention in ASIC1a mediated neuronal death by targeting NSF. Using NSF knockdown shRNA and its inhibitor NEM (N-ethylmaleimide), it was found that intervention NSF can act on ASIC1a-RIPK1 programmed death signaling pathway, affect dissociation at both ends of N C, and have protective effects in an acid-toxin mediated cell death model. NSF was specifically involved in ASIC1a mediated neuronal death in acidosis (fig. 5).

In fig. 5, (a) Western blot verifies the knockdown efficiency of NSF shRNA against NSF protein, which was designed against a sequence of mouse NSF (GCTTCAATGATAAGCTCTT). (b) (a) summarizing the data. (c) NSF knockdown can reduce binding of ASIC1a to RIPK 1. (d) And (c) summarizing data by the graph. (e) NSF knockdown can affect the dissociation of ASIC1a N-and C-terminals. (f) (e) summarizing the data by the graph. (g) PI staining of wild-type mouse cultured cortical neurons showed that NSF knockdown reduced acidification-mediated neuronal death, whereas there was no significant difference between the knockdown NSF in ASIC1a knockout mouse cultured cortical neurons and the control group, suggesting that NSF is specifically involved in ASIC1 a-mediated neuronal death in acidosis. (h) (g) the graphs summarize the data. (i) NSF inhibitors have a protective effect in models of acid-toxin mediated cell death.

Example 6 glutamic acids 6 to 9 distal to ASIC1a N were critical for the formation of intramolecular self-inhibition by ASIC1a

The 6 th to 9 th glutamic acid at the N terminal of ASIC1a is predicted to be a key amino acid site for forming self-inhibition by computer simulation. After mutation of glutamate to alanine, N C ends are at an increased distance, basal cell death increases and the programmed necrosis pathway is activated. Mixing NT_1-20NT with mutation of glutamic acid from 6 th to 9 th positions in polypeptide into alanine_1-20 ^E/AThe protection of the polypeptide in the acidosis and cerebral ischemia model disappeared (fig. 6).

In FIG. 6, (a) the closed ASIC1a single subunit Rosetta modeling indicates that the N-terminal is negatively charged6 to 9 glutamic acids E⁶EEE⁹Closely aligned with the positively charged amino acid sites of K468, K471 and K474 in the C-terminal toxic fragment, the distance between the beta carbon atoms in E7 and K468 was approximately 4.7A. The distance between the two increases after mutation of glutamate E to alanine a. (b) The FRET value of the wild plasmid is reduced under the basic state after 6 th-9 th amino acid glutamic acid is mutated into alanine, which indicates that the distance between the N end and the C end of the ASIC1a is increased under the basic condition after mutation. (c) PI staining shows that transfection of the E/A mutant plasmid on CHO cells can result in increased cell death in basal cases. (d) And (c) summarizing data by the graph. (e) The co-immunoprecipitation results showed that the E/A mutant had increased binding of ASIC1a to NSF, ASIC1a to RIPK1 at pH 7.4 base. (f, g) (e) the graphs summarize the data. (h) Evaluation of neuronal death results by lactate dehydrogenase Release amount indicates NT_1-20Can mediate acid toxicity neuroprotection to a similar degree as that of Nec-1 which is an inhibitor of RIPK1, however, after the glutamic acid site of the polypeptide is mutated into alanine, the protection effect of the polypeptide in an ex vivo acid toxicity model disappears. (i) TTC staining showed NT_1-20The polypeptide can effectively reduce the infarct volume caused by cerebral artery embolism of mice, and the protection effect of the polypeptide disappears after the glutamic acid site is mutated into alanine. (j) (i) the graphs summarize the data.

In summary, the working model of the ASIC1a proposed by the present invention is: as shown in fig. 7, under normal physiological conditions, the N-terminus of ASIC1a sequesters the toxic C-terminus, forming intramolecular self-inhibition (left). In the event of ischemic stroke, a large amount of acidic material is accumulated in the tissue, acidosis induces intracellular NSF to bind to the N-terminal of ASIC1a and activates ASIC1a causing its conformational change, exposing the C-terminal lethal sequence, the C-terminal toxic fragment binds to RIPK1, which in turn undergoes phosphorylation, mediating programmed cell necrosis (middle). When NT is added_1-20When the protective peptide is used, the protective peptide is combined with the C end of ASIC1a, so that the protective peptide inhibits the recruitment and activation of RIPK1, and plays a role in neuroprotection. At the same time, NT_1-20The exogenous fragment also competitively binds to intracellular NSF, so that the N-terminal cannot be dissociated from the C-terminal and the toxic fragment at the C-terminal cannot be exposed, so that the cells survive and are protected from damage caused by stroke, and the effect is equal to that of using NSF to knock down shRNA and using NSFInhibitor NEM (right).

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Claims (4)

1. A neuroprotective agent, wherein the neuroprotective agent is a transmembrane protective peptide mimicking the N-terminus of ASIC1a, the transmembrane protective peptide having an amino acid sequence shown in SEQ ID No. 1.

2. A pharmaceutical composition for treating and/or preventing a cerebral stroke disease comprising a safe and effective amount of the neuroprotective agent of claim 1, and a pharmaceutically acceptable carrier.

3. Use of the neuroprotective agent according to claim 1 for the preparation of a medicament for the treatment and/or prevention of cerebral stroke diseases.

4. Use of a neuroprotective agent as claimed in claim 1 in combination with at least one thrombolytic agent for the manufacture of a medicament for the treatment of stroke disorders.

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