CN117417914A - Marker for preventing and protecting cerebral ischemia diseases and application of aspirin derivative in preventing and protecting cerebral ischemia diseases - Google Patents

Marker for preventing and protecting cerebral ischemia diseases and application of aspirin derivative in preventing and protecting cerebral ischemia diseases Download PDF

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CN117417914A
CN117417914A CN202210840260.4A CN202210840260A CN117417914A CN 117417914 A CN117417914 A CN 117417914A CN 202210840260 A CN202210840260 A CN 202210840260A CN 117417914 A CN117417914 A CN 117417914A
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aspirin
alpha
acetylation
camk2
site
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王碧含
韩威
殷东敏
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East China Normal University
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Abstract

The invention discloses a novel molecular target for aspirin to play a role in neuroprotection on cerebral apoplexy. Aspirin exerts neuroprotective effects on stroke-model mice by acetylation of the intracellular calcium ion-calmodulin dependent protein kinase 2α (CaMK 2 α). The invention clarifies a new mechanism of aspirin to exert neuroprotective effect independent of its anticoagulant function. The invention also discloses that aspirin derivatives can also exert neuroprotective effect on mice with cerebral apoplexy types through acetylation of CaMK2 alpha, but the anticoagulation effect and bleeding risk of the aspirin derivatives are weaker than those of aspirin. The invention also discloses a safe and effective new strategy for preventing cerebral apoplexy: through modifying aspirin, the anticoagulant activity of aspirin is reduced, but the function of the aspirin in the acetylation of CaMK2 alpha is reserved, so that the neuroprotection effect of preventing cerebral apoplexy can be better exerted, and the side effect of bleeding is reduced. The invention provides a safer and more effective way for the preventive medication of cerebral ischemia diseases.

Description

Marker for preventing and protecting cerebral ischemia diseases and application of aspirin derivative in preventing and protecting cerebral ischemia diseases
Technical Field
The invention belongs to the technical field of medicines, discloses a novel molecular target point of aspirin for preventing cerebral apoplexy, clarifies a novel mechanism of aspirin cerebral ischemia protection without depending on anticoagulation, develops novel aspirin derivatives on the basis, and can more safely and effectively prevent cerebral apoplexy.
Background
In every 4 people worldwide, 1 person can generate cerebral apoplexy (commonly called cerebral apoplexy or cerebral ischemia), and every 6 seconds, 1 person dies from the cerebral apoplexy. Cerebral apoplexy has become the "first killer" threatening human life health and quality of life because of its high prevalence, high recurrence rate, high mortality and disability rate. According to the Chinese cardiovascular disease health and disease report 2019 issued by the Chinese cardiovascular disease center, the number of cardiovascular patients in China is up to 3.30 hundred million, and 1300 ten thousand patients suffering from cerebral apoplexy are among them. The world health organization indicates that daily oral administration of aspirin can effectively prevent the occurrence of cerebral stroke through anticoagulation. Aspirin treatment is helpful for people at risk of cerebral apoplexy (such as people with history of cerebral apoplexy). However, aspirin often brings side effects such as bleeding risk and gastric mucosal injury during the use process. Therefore, a new mechanism independent of anticoagulation is needed to be explored for the neuroprotection of aspirin, and aspirin derivatives are developed through the new mechanism, so that the aspirin derivatives exert cerebral ischemia protecting effect and reduce bleeding side effects, thereby providing a better way for preventive administration of heart cerebral apoplexy.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a novel molecular target point for the aspirin to play a role in neuroprotection on cerebral apoplexy for the first time in the process of researching the aspirin to play the role in neuroprotection on cerebral apoplexy. Aspirin is produced by acetylation of the calcium ion-calmodulin dependent protein kinase 2 alpha (Ca 2+ Calm module-dependent kinase 2. Alpha., caMK 2. Alpha.) exert neuroprotective effects on mice of the cerebral apoplexy type. The invention firstly clarifies that aspirin plays a role in neuroprotection independent of aspirinA novel mechanism of anticoagulant function.
The invention also provides a safe and effective new strategy for preventing cerebral apoplexy and cerebral ischemia diseases: through modifying aspirin, the anticoagulant activity of aspirin is reduced, but the function of the aspirin in the acetylation of CaMK2 alpha is reserved, so that the neuroprotection effect of preventing cerebral apoplexy can be better exerted, and meanwhile, the side effect of bleeding is reduced. The invention provides a safer and more effective way for preventive medication of cerebral apoplexy and cerebral ischemia diseases.
The present invention proposes a new strategy for improving aspirin to increase its benefit-risk ratio, and is not limited to the aspirin derivative, ethyl acetylsalicylate. Other aspirin derivatives meet the following two requirements: 1) Weakening the binding force with COX-1 and reducing the anticoagulation function; 2) The ability to acetylate the CaMK2 alpha protein in the brain is preserved; can function like ethyl acetylsalicylate.
The chemical formula of the aspirin is C 9 H 8 O 4 The molecular structural formula is CH 3 COOC 6 H 4 COOH, molecular relative mass 180.16, CAS number 50-78-2.
The invention provides a biomarker which is an acetylation site of calmodulin-dependent protein kinase 2 alpha (CaMK 2 alpha) in nerve cells.
The amino acid sequence of CaMK2 alpha is shown in SEQ ID NO. 1:
matitctrft eeyqlfeelg kgafsvvrrc vkvlagqeya akiintkkls ardhqklerearicrllkhp nivrlhdsis eeghhylifd lvtggelfed ivareyysea dashciqqileavlhchqmg vvhrdlkpen lllasklkga avkladfgla ievegeqqaw fgfagtpgylspevlrkdpy gkpvdlwacg vilyillvgy ppfwdedqhr lyqqikagay dfpspewdtvtpeakdlink mltinpskri taaealkhpw ishrstvasc mhrqetvdcl kkfnarrklkgailttmlat rnfsggksgg nkkndgvkes sestnttied edtkvrkqei ikvteqlieaisngdfesyt kmcdpgmtaf epealgnlve gldfhrfyfe nlwsrnskpv httilnphihlmgdesacia yiritqylda ggiprtaqse etrvwhrrdg kwqivhfhrs gapsvlph
the nucleotide sequence is shown in SEQ ID NO. 2:
agaagcccca agctcgtcag tcaagccggt tctccgtttg cactcaggag cacgggcaggcgagtggccc ctagttctgggggcagcgct tcagcatccc agccctagtt cccagcctaaagcctcgcct gcctgcccag tgccaggatg gctaccatca cctgcacccg attcacagaagagtaccagc tctttgagga actgggaaag ggagccttct ctgtggtgcg caggtgtgtg aaggtgctgg ctggtcagga gtatgctgcc aagattatca acaccaagaa gctttcagccagagatcacc agaagctgga gcgtgaggcc cgcatctgcc gcttgttgaa gcaccccaatatcgtccgac tccatgacag catctccgag gaggggcacc actaccttat cttcgatctggttactggtg gggaactgtt tgaagacatt gtggcccggg agtattacag tgaagctgatgccagccact gtatccagca gatcttggag gctgtgctac actgtcacca gatgggggtggtgcatcgtg acctgaagcc tgagaatctg ttgctggcct cgaagctcaa gggcgctgctgtgaagctgg cagactttgg cctggccata gaggtggagg gggagcagca ggcatggtttgggttcgcag ggacacctgg atacctctcc ccagaagtgc tgaggaagga cccgtacgggaagcccgtgg acctgtgggc ctgtggcgtc atcctgtata tcttgctggt tgggtatcccccgttctggg atgaagacca gcatcgcctg taccagcaga tcaaagctgg tgcctatgat ttcccatcac cagaatggga caccgtcacc ccagaagcca aggatctgat caataagatgctgaccatca acccgtccaa acgcatcacg gccgctgagg ctctcaagca cccatggatctcgcaccgct ccaccgtggc ctcctgcatg cacagacagg agaccgtgga ctgcctgaagaagttcaatg ccaggaggaa actgaaggga gccatcctca ccactatgct ggccaccagg aacttctccg gagggaagag cggaggaaac aagaagaacg atggtgtgaa ggaatcttctgagagcacca acaccaccat tgaggacgaa gacaccaaag tgcgcaaaca ggaaattatcaaagtgacag agcagctgat cgaagccata agcaatggag actttgagtc ctacacgaagatgtgcgacc ctggaatgac agcctttgaa ccagaggccc tggggaacct ggtggagggc ctggactttc atcgattcta ttttgaaaac ctgtggtccc ggaacagcaa gcccgtgcacaccaccatcc tgaaccctca catccacctg atgggtgacg agtcagcctg catcgcctatatccgcatca ctcagtacct ggatgcaggc ggcatacccc gcacggccca gtcagaggagacccgcgtct ggcaccgcag ggacggcaaa tggcagatcg tccacttcca cagatctggg gcgccctccg tcctgccgca ttgaaggacc aggccagggt ccctgcgtcc ttgcttcgcagagatccgct ctttgtccgt ggaatgtggc tgctggttct cctttggatt ttgctggaattctccctgtc agatcaccct accattgcca cctatgtact cgcgtcacga aaacctgctt gttcacagaa gtcgccacga catcacagtg aacagccagc tctccccagc tccgttgcccaagctcttcc tgccagtggg gaccttcttc cggcttaagt acccagggtg ctggccccaggaacccccac cccctaccca ctgttgttgg cctagcctag ctttagctat agatggggcc tcagctgtgc aattggcagg aagtgaggaa gaggcaggca agctgtgttg agggcacctctcatcgattc cttctttcct ggggttcccc ggggaagctc acacgaggcc ctcagtctccaagccaaccc cttatgaggg agagtgagag aggagccaac gccagtgagc caggaactgctgctctcatc tgctctcctc tgtgttggcc ttgcctttga ccagaccatc cgctacgaggggtgggctct accgcccagg tgccccactc actctgcctc agtcctcctg tgaagtttgcctccagtgtt gacccaccca ccctgccctt caacgtcctt ggagaattcc agcttcatctgtctgagagg agattggaag gtgtttcagg ggcaaagcaa gcaacattta ggtatcacttctacttggac gcatgccttt ttacagccaa acttccgtgt atttcgtaaa tggattttgcgttaacggac atctatgtga taactagacc tctcaagttt actgtaaaga agagtcggatgggttgggaa gtgggtggga agaggagtga gaggaagttt taaccccatt ccagagggtcttttttttgg ggggtgtccc ttctggggga gggtgctttc tgaggtggcc tcaccccccagggagcatgg tttccttcca ttatggtccc caaagtcagc tgacaagatt tcttccagagccagcatgac taacacacag tgagtcaggt caggggaggc tatcaggaag gggttatctagacttggcat ctctggaact ggagacctca ctaccctact tcccaggaaa tctttcccgtatccttgtgc tatctctcct cagcccagcc cccactcacc accttctagg cacattattctcatcacctc tctcaacttg gccaagcaag agtgccagaa acttattttc ctacttcatgctgaacttgg cctggtgtgc gctctattgt gcttgcctgc ctccctggcc ttcctcgactcccctgcccc gacatacaca cacacacaca cacacacaca cacacgcaca cgcacacgcgcgcacacaca cacatgcaca cgcacatgca cacactcaca ccacttcctt ccaccacttccttcctcccc cctctccctc cctttctctt tctcttctct ccctcttcct gggtttggctcttgtatgga atgctgtatc tcattcacgg ggatctcctg tctgcactgt tttctttgca tgactttata tgcagtaagt atgttgaaaa acaaacaaaa aacaaaaaag gagaaaaacactcaacaaaa tcaaacgaca cgtttttttt tggacaaaaa ataataacat tcaaggtaatagtctcagtg tccaacttgg acttacgttg ctgcctctcc gtgcttttgg tctctctgtg gctatgtttt gccagcatga gaccctgttc cctctggaag ttgctagggg aggaagagccatgcgtccag gggtttggag acagctttat cctctcggct tttctgaggg tcgatgggagcagaagtgga agggatgttt aatccagaac tttctggtat tcccctttcg cctacatgtg agctatatcc cgggctcttc tctcaaatcc tgctgcccag ggacaagtac agggtagaagagtggctctc ttgtctaagc cgctccactg tagcctctgc ccttggtaga gacactgctacccaggccca agaatgggcc cttgtcctgc cccagaacca ggcttcttca taaggctcag caaacccatt gtccccagcc actccccaga taaaggtaaa ggagggtgtg gcctttaccaggggacactg caatctccat gcaggatcta caatcccttc ttcaagcctc agtttctccatcaatgttcc tacccagact gatggagggt cagagtaaaa gatgtcacaa gcacccacca cctctgagag cttgttgggt ttgtcactgg ctggccctct tatgcaccag gtctggccaacccccaccct ttcctctgtg tgcccctcat tttactattt ggtgccagtc cgttgatgaccagcaatgga ctgcagggga aagaagtgtt tgggggctct gatcccgggt tctgaccaga tccgggtttt gtgtagcttt gggtaaatcc tttgccctct tcaggcttta gtttcgccaaccagaagatg cctatgccct gccttccgtt ggctaacatg cccctgtcca ctatgtgcttgttcacgtgt gggagaagtg gaggcaagtc cctgccccag tctgagacgg ccccctctgc agaggccgct cctgtgggtg ggcagccaac taatgaagac cttgggacac tacgatggccccaaggtgac aggcagggga acaggcagaa aaactgtcca gagcccaccc tcatctgacaagctccatgc tccgtccaaa taccctccag atgaaaaaaa aaagagagag agagagaaagaaacaaagag tcaaatcaca tttataggaa aagcgtctcc agctctatgc accatagctc aaatcctgcc ccatggcttc cccacccccc ttcaaaggga gagccttggg ggaatgcgtttgccaggccc cgtgctggct tctttgttac tatttgttta gggttttgtt ctagttctctctctctctct ctctctctct ctctctctct ctctctctct ctctttcttt tttaatctgtggctgtgaac ttgaatgacc actgctcaaa ctttctgcta ctgggggggt gggggaggggagaagagatg tctggtttat tcttggcgtt ttcagtggaa taaatagcta caaatttatgtgagtccgtg tcttcctgaa ttggtcaagg cacagagccc caggaactgg cattttgctttggcttgttt tttgggtttt ttgtttgttt gtttgttttt gtttgtttca aatctcccctgttgcaaaat aaaagtcctg gtcctatgga ttggaaaaaa aaaaaa。
the acetylation sites are K42 and K286.
The invention also provides application of the biomarker in preparing a medicine for preventing and/or treating cerebral apoplexy and cerebral ischemia.
In the application of the invention, the medicine is aspirin and/or aspirin derivatives, modifications and modified substances.
In the application of the invention, the medicine comprises aspirin derivative ethyl acetylsalicylate.
The chemical formula of the aspirin derivative acetylsalicylic acid ethyl ester is C 11 H 12 O 4 The molecular weight is 208.21100, and the product number is 529-68-0.
In the application of the invention, the aspirin increases the acetylation level of CaMK2 alpha by acetylating protein kinase 2 alpha (CaMK 2 alpha) dependent on calcium ion-calmodulin in nerve cells, inhibits the kinase activity (autophosphorylation and substrate phosphorylation) of the CaMK2 alpha, thereby affecting the downstream protein of the CaMK2 alpha to exert corresponding effects and generating direct neuroprotection.
In the application of the invention, the aspirin increases the acetylation level of the K42 site of the CaMK2 alpha in the brain by about six times (the acetylation modification level of the K42 site of the CaMK2 alpha is continuously increased from about 0.5% of the normal condition and is stable at 3%).
In the application of the invention, the aspirin derivative acetylsalicylic acid ethyl ester, the modifier and the remodelling substance can increase the acetylation level of the CaMK2 alpha through acetylating the protein kinase 2 alpha (CaMK 2 alpha) dependent on calcium ion-calmodulin in nerve cells, inhibit the kinase activity (autophosphorylation and substrate phosphorylation) of the CaMK2 alpha, thereby affecting the downstream protein of the CaMK2 alpha to play a corresponding role and generating a direct neuroprotective effect.
In the application of the invention, the calmodulin-dependent protein kinase 2 alpha (CaMK 2 alpha) is a calmodulin kinase family, and can regulate the activity and the function of various protein types expressed upstream proteins, and the activity of the upstream proteins is related to the neuroprotective function.
The camk2α of the present invention consists of 28 similar isoforms from 4 genes (α, β, γ and δ), the α -and β -subunits being the major subtypes in the brain, forming a dodecamerase consisting of one or two subunit types.
In the application of the invention, the acting site of the aspirin capable of acetylating the calmodulin-dependent protein kinase 2 alpha (CaMK 2 alpha) is a K42 (Lys 42) site and a K286 (Lys 286) site.
In the application of the invention, the action site of the aspirin derivative acetylsalicylic acid ethyl ester, a modifier and an remodelling substance capable of acetylating calcium ion-calmodulin dependent protein kinase 2 alpha (CaMK 2 alpha) is a K42 (Lys 42) site.
Lys42 refers to lysine 42 marked by the Ballesteros-Weinstein numbering method in the amino acid sequence of CaMK2 alpha.
Lys286 refers to lysine 286 marked by the Ballesteros-Weinstein numbering method in the amino acid sequence of CaMK2 alpha.
In the use of the invention, the acetylation of lysine number 42 inhibits camk2α activity by interfering with binding to ATP rather than calcium binding.
In the application of the invention, the change of the CaMK2 alpha activity is measured by using a kinase kit.
In the application of the invention, the acetylation modification of the K42 locus of CaMK2 alpha is enriched in cytoplasm (the ratio of the acetylation protein to the total protein is up to 12%), and the ratio of the acetylation modification in synapses is lower (the ratio of the acetylation protein to the total protein is only 2%).
In the use of the invention, the calmodulin-dependent protein kinase 2 alpha (CaMK 2 alpha) is capable of autophosphorylation and phosphorylation of a substrate, the activity of which is associated with neuroprotective function.
In the present application, the acetylation of lysine number 42 inhibits the kinase activity of camk2α by interfering with ATP binding rather than calcium binding.
In the application of the invention, the K42 site is an ATP binding site of calmodulin kinase, and acetylation modification of the K42 site obviously damages the binding of CaMK2 alpha protein kinase and ATP.
In the application of the invention, the occurrence of the acetylation modification of the lysine K42 site does not affect CaMK2 alpha and Ca 2 + Binding between/CaM.
In the application of the invention, the acetylation of the K42 site of CaMK2 alpha can inhibit autophosphorylation and phosphorylation of a substrate, but the acetylation of the K258 site does not affect the autophosphorylation of CaMK2 alpha and the phosphorylation of the substrate.
Among the applications of the invention, the application includes the application of aspirin and/or aspirin derivatives, modifications and reforms in preparing drugs for preventing and/or treating cerebral apoplexy diseases and recurrent cerebral ischemia, the application in preparing drugs for patients who cannot use/cannot use aspirin in large dosage but have cerebral ischemia risks at the same time, or the application in preparing drugs for substitution therapy of aspirin.
The aspirin derivative ethyl acetylsalicylate maintains the effect of aspirin in preventing cerebral ischemia, reduces the bleeding risk of aspirin, and improves the safety of cerebral ischemia prevention medicines.
In the application of the invention, the effectiveness of the aspirin derivative ethyl acetylsalicylate as a medicament for preparing the medicament for preventing/treating recurrent cerebral ischemia adopts a method which is compared with aspirin, and the aspirin is evaluated and judged according to biochemical experimental indexes of the aspirin and the aspirin, which are provided by the invention, and plays a direct mechanism of neuroprotection in preventing cerebral ischemia related diseases.
In the application of the invention, the safety of the aspirin derivative ethyl acetylsalicylate as a medicament for preparing the medicament for preventing/treating recurrent cerebral ischemia adopts a method which is compared with aspirin, and the safety is based on the experimental model animal blood coagulation function test.
The invention also provides a regulator of the biomarker, which comprises an agonist, an inhibitor or an antagonist of the biomarker.
The invention also proposes a pharmaceutical/pharmaceutical composition comprising said biomarker, or comprising a compound that can also bind to or react with said biomarker.
Wherein the medicine/pharmaceutical composition comprises aspirin and/or aspirin derivatives, modifications and modified substances.
Further, the drug/pharmaceutical composition comprises the sappan derivative ethyl acetylsalicylate.
The invention also provides a detection reagent/kit comprising an active ingredient useful for detecting a biomarker as described above and its level of acetylation.
The invention also provides a polyclonal antibody specifically recognizing the acetylation CaMK2 alpha at the K42 locus, which is prepared by selecting 37 th to 48 th amino acid synthesis modified polypeptide (C-QEYYAK Ac IINTKK) coupleKLH (Keyhole Limpet Hemocyanin, hemocyanin) in combination, obtained after immunization of rabbits; the antibody can specifically recognize the CaMK2 alpha protein acetylated at the K42 site, can not recognize the wild non-acetylated CaMK2 alpha protein and can not recognize the CaMK2 alpha protein acetylated at other sites such as K258.
The invention also provides a preparation method of the acetylation modified polyclonal antibody specifically recognizing CaMK2 alpha, which comprises the following steps:
(1) Specific acetylation modification of K42 site of CaMK2 alpha, selecting 37 th to 48 th amino acid synthesis modified polypeptide (C-QEYYAK Ac IINTKK), coupled to KLH (Keyhole Limpet Hemocyanin, hemocyanin), immunized into rabbits, sacrificed, serum purified antibodies, and control peptide (C-QEYYAKIINTKK) synthesized, purified and detected by enzyme-linked immunosorbent assay (ELISA);
(2) Protein (His-K42) modified by site-directed acetylation of whole K42 site is directly synthesized and purified in vitro by utilizing non-natural coding acetylated lysine technology Ac CaMK2α) and K258 site-directed acetylation modified protein (His-K258) Ac -CaMK2α)；
(3) The purity of the protein was detected by coomassie brilliant blue staining, and then the specificity of the antibody in vitro was detected by using purified site-directed acetylated protein, by constantly optimizing conditions and performing multiple purifications of the antibody.
The invention also provides a method for preparing the acetylated CaMK2 alpha protein, which comprises the step of site-directed acetylation of modified protein (His-K42) with full K42 site Ac CaMK2α) and K258 site-directed acetylation modified protein (His-K258) Ac -CaMK2α)。
The invention also provides a method for preparing the acetylated CaMK2 alpha protein, which specifically comprises the following steps:
in vitro purification of site-directed acetylated proteins (His-K42) AC -CaMK2α、His-K258 AC -CaMK2α)
List of plasmids:
pET-N-His-CaMK2α
pET-N-His-K42TAG-CaMK2α
pET-N-His-K258TAG-CaMK2α
pCDF-AcKR3-tRNACUA
primer design details:
point mutation primer design
Escherichia coli purified protein (His-K42) AC -CaMK2α、His-K258 AC -CaMK2α)：
(1) Transformation of competent E.coli cells
Resuscitating: rapidly taking out (expression competent cells) BL21 from-80deg.C refrigerator, and standing on ice for 10min for cell resuscitation;
conversion: adding 5 μl of mutant plasmid into 50-100 μl of competent cells after resuscitating, and standing on ice for 20-30min;
heat shock: heat-beating in a water bath at 42 ℃ for 50-90s, immediately putting back on ice and standing for 2min; adding 900-1000 mu l of antibiotic-free LB liquid culture medium, shaking and culturing at 37 ℃ and 200rpm for 1h by a constant-temperature shaking table to enable bacteria to grow normally; to increase the transformation efficiency of the mutant, the mutant is centrifuged at 3000rpm for 1min at normal temperature, 600 μl of culture medium is sucked off in a sterile environment, resuspended thalli are blown off slowly, and about 100 μl of coated plates are sucked;
the bacterial liquid is coated on LB solid culture medium plates with corresponding resistance, and the incubator is inverted and cultured overnight at 37 ℃.
(2) Transformation of competent E.coli cells
Small shake: the monoclonal colonies were picked up from the medium with a picking needle, and the colonies were directly placed into a liquid medium (containing the corresponding antibiotics as plasmid vectors) containing 5ml for cultivation, and the incubator 200 was rotated overnight at 37 ℃.
Large shaking: when the medium with small shaking becomes turbid, the bacterial cells are propagated and cultivated on a large scale, and then all the bacterial liquid with small shaking is directly transferred into 500ml of liquid medium for cultivation. The density of the large shaking thalli needs to be controlled to be between 0.4 and 0.6 in an oven with the temperature of 37 ℃ and 200 revolutions.
Induction of expression: when the OD value of the large shaking bacterial liquid reaches the interval of 0.4-0.6, the bacterial liquid is taken out, 0.5mmol/L IPTG is added into an ultra-clean bench for induction expression, and the bacterial liquid is put into an oven again for induction expression for 6h. Since the site-directed acetylated protein was purified, 1mM of acetylated lysine was additionally added, and after half an hour of incubation, the inhibitor NAM of deacetylase was added and incubation continued for 6 hours.
Cleavage of proteins: after 6h of induced expression, the bacterial liquid is taken out, centrifuged, the supernatant is removed, and the bottom bacterial cells are reserved. And (3) putting the thalli into ice for cracking by using thalli lysate, and performing ultrasonic cracking, wherein each ultrasonic process lasts for 10s, and the interval between the two ultrasonic processes is 5min. After about 5 to 10 times of ultrasound, it is observed whether or not the cells become permeable, and if not, the cells may be added in an excessive amount without sufficient lysis, and the number of ultrasound may be increased appropriately.
Purifying protein: the cells lysed on ice were removed, centrifuged for 12000 g.times.15 min, and the supernatant was retained to remove the sediment on the lower layer. Protein concentration was determined by BCA method, 5-10 mg of protein cell lysate was taken per tube, diluted to 1ml with PBS, then 100ul of His-beads were added per tube, and after shaking suspension, the suspension was placed on a rotary suspension apparatus for overnight suspension at 4 ℃. The next day, the suspended tube was removed, centrifuged for 1500g×3min, and the supernatant was removed to retain the lower beads, and eluted with PBS for 6 consecutive times at 1500g×3 min. After elution, 200ul of protein eluent is added into each tube of magnetic beads, the mixture is blown and suspended for 60s, and the mixture is put into a centrifuge 10000g multiplied by 10min for centrifugation, the eluent on the upper layer is reserved, and the magnetic beads on the lower layer are removed. Protein concentration determination was performed by a spectrophotometer. Packaging, and storing in refrigerator at-80deg.C.
The invention also provides application of the biomarker regulator, the medicine/medicine composition, the detection reagent/kit or the antibody/protein in predicting, evaluating, preventing and/or treating cerebral apoplexy diseases, cerebral ischemia diseases and recurrent cerebral ischemia diseases, in preparing medicines for patients who cannot use/cannot use aspirin in large dosage but are at risk of cerebral ischemia at the same time, or in preparing medicines for substitution treatment of aspirin.
In the application of the invention, the regulator, the medicine/medicine composition and the detection reagent/kit can be used singly or in combination with other medicines.
The invention also provides a medicine transformation thought for preventing/treating cerebral ischemia diseases, and acetylation of CaMK2 alpha lysine 42 locus can be used as a new target for preventing/treating cerebral ischemia diseases.
The invention also provides an action mechanism of the medicine or the medicine combination in preventing/treating cerebral ischemia diseases, and measurement and evaluation of safety and effectiveness.
The invention also provides a method of screening for a candidate drug for the prevention/treatment of cerebral ischemic disease, the method comprising detecting an effect of the candidate drug on a biological effect of the biomarker in a subject or a sample obtained from the subject, comprising detecting an effect of the candidate drug on the acetylation level of position 42 of camk2α or the kinase activity of camk2α or a biological effect downstream of its signal in the subject or the sample obtained from the subject.
The invention also provides a method for preventing or treating cerebral apoplexy and cerebral ischemia related diseases of a subject in need thereof, or a method for predicting or prognosis evaluating whether aspirin/aspirin derivatives, modifications and alterations are applicable to the patient for preventing/treating cerebral apoplexy and cerebral ischemia related diseases.
The method comprises detecting the level of acetylation at the K42 site of camk2α or the kinase activity of camk2α of a biomarker as described above.
The aspirin derivative ethyl acetylsalicylate can reduce the bleeding risk relative to aspirin, and a coagulation experiment detects and compares the coagulation time of mice after 14 days of feeding aspirin and ethyl acetylsalicylate, and the total coagulation time is found to be increased by aspirin, and the coagulation time is obviously reduced by the ethyl acetylsalicylate.
The aspirin derivative ethyl acetylsalicylate maintains the capacity of acetylation of the K42 locus of CaMK2 alpha protein, and compared with aspirin, the ethyl acetylsalicylate can better improve the acetylation level of CaMK2 alpha.
Both aspirin and the aspirin derivative ethyl acetylsalicylate exert neuroprotective effects independent of their anticoagulant function.
The innovation point of the invention is that a new mechanism of the aspirin for exerting the neuroprotection on cerebral apoplexy is provided, which is not dependent on the anticoagulation function of the aspirin, and the aspirin derivative, namely the acetylsalicylic acid ethyl ester, has better benefit-risk ratio in preventing and protecting cerebral ischemia diseases compared with the aspirin, that is, the bleeding risk of the aspirin derivative, namely the acetylsalicylic acid ethyl ester, is smaller than that of the aspirin, and the neuroprotection is equivalent to that of the aspirin.
The invention has the beneficial effects that: in the process of deeply researching the neuroprotection of aspirin to cerebral apoplexy, the invention provides a novel molecular target point of aspirin for protecting cerebral apoplexy. Aspirin exerts neuroprotective effects on mice of the stroke type by acetylation of the calcium ion-calmodulin dependent protein kinase 2 alpha within the nerve cells. This finding illustrates a new mechanism by which aspirin exerts neuroprotective effects independent of its anticoagulant function. The invention reduces the anticoagulant activity of aspirin by modifying the aspirin, but retains the function of the acetylated CaMK2 alpha, can better play the neuroprotection role of preventing cerebral apoplexy, and reduces the side effect of bleeding. The invention provides a safer and more effective way for the preventive medication of cerebral ischemia diseases, and has wide application prospect in the field of the prevention/treatment of cerebral ischemia diseases.
Drawings
FIG. 1 is a protein profile identifying aspirin-regulated mouse brain cortex acetylated proteins and lysine sites. Aspirin can directly increase the acetylation level of camk2α in a dose-dependent manner, and K42 and K258 are the main acetylation sites.
FIG. 2 is a schematic illustration of the preparation and validation of a rabbit polyclonal antibody specifically recognizing an acetylation modification at the CaMK 2. Alpha.K42 site.
FIG. 3 quantification of protein acetylation modification occurrence in the mouse brain.
FIG. 4 acetylation of the K42 site by interferenceBinding to ATP but not interfering with Ca 2+ CaM binding inhibits CaMK2 alpha kinase activity.
FIG. 5 interferes with acetylation of the CaMK 2. Alpha. Protein K42 site to impair aspirin protection in ischemic stroke mice models.
FIG. 6 shows that the expression of the K42 site acetylated CaMK 2. Alpha. In eukaryotic systems, and that the expression of the K42 site acetylated CaMK 2. Alpha. Protein in the mouse brain has direct neuroprotection.
FIG. 7Asp acetylated CaMK 2. Alpha. At the K42 site, binding between CaMK 2. Alpha. And ATP was blocked, kinase activity was inhibited and K42 acetylated CaMK 2. Alpha. Was mainly enriched in the cytosol.
Fig. 8 ethyl acetylsalicylate performs better in neuroprotection than ASP, while reducing bleeding risk.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited.
Example 1
(1) In vivo experiments
1) Experimental animals: clean grade male wild type mice (C57 BL/6), 72, 8-12 weeks old; c57BL/6 mice of Camk2α -K42R strain, 6 mice, 8-12 weeks old; c57BL/6 mice of Camk2α -knock out strain, 6, 8-12 weeks old. All mice were free-drinking and maintained at 12h:12h light: in a dark constant temperature and humidity environment. Mice were grouped into (1) control group: clean grade male wild mice were fed ultrapure water; (2) aspirin group: clean male wild type mice were fed water (Asp, 0.1 mg/mL) containing aspirin at a concentration; (3) group of ethyl acetylsalicylates: feeding clean grade male wild mice with water (0.1 mg/mL) containing a concentration of ethyl acetylsalicylate groups;
2) Construction of an MCAO animal model: the MCAO animal model construction experimental procedure refers to the Longa modified line plug method for establishing a mouse focal cerebral ischemia model, and the method is accepted by the global academy. Before the operation, the operation is carried out,the mice were arranged in a 12:12 light/dark cycle environment with free access to water and rodent chow. Surgical procedure: after weighing the mice, anesthesia was induced in the induction chamber with 4% isoflurane, and during the course of the treatment with 1.5-2% isoflurane in 30% O 2 And 70% N 2 Is maintained under anesthesia by the mask. Placing a mouse in a prone position on a gauze pad placed on a temperature-controlled operating surface (heat blanket); the hair on the head is shaved. During the operation, the eyes are coated with lubricant eye ointment, and the eyes are closed to prevent the eyes from drying. The neck of the mouse is placed on a cushion and its nose is secured in the nose cone of the anesthesia apparatus. Fixing the mouse in supine position, sterilizing neck skin with 75% alcohol, slightly right 1cm incision in the middle of neck, separating subcutaneous muscle and tissue layer by layer, separating common carotid artery, internal carotid artery and external carotid artery, ligating common carotid artery and external carotid artery, opening 1 small hole at 1.5cm distance between common carotid artery and bifurcation, and slowly inserting the plug with silica gel in the head end until the plug is slightly resistant. The insertion depth is about 9-10 mm (from the bifurcation between the inside and outside of the neck), causing the middle artery on the right side of the brain to be blocked, resulting in cerebral ischemia. The thread plug is fixed, and the muscle and the skin are sutured layer by layer. After 1h of ischemia, the tether was pulled out and cerebral ischemia reperfusion was performed. Finally, the mice were removed from the surgical sheath and placed in a warm cage to prevent hypothermia and to have sufficient, readily available soft food and water.
3) Administration: the water containing the medicine in the mouse drinking bottle is replaced periodically, so that the medicine in the mouse drinking bottle is prevented from being degraded;
4) Sample collection: after the treatment according to different experiments is finished, the head of the mouse is sheared off, the skin of the head is sheared off, and the exposed skull of the tissue is cleaned; the scalp bone is uncovered from the hindbrain to the front, the whole brain is exposed, the whole brain is carefully held out by using a fine bending forceps, and the whole brain is washed clean in normal saline and stored in a refrigerator at the temperature of minus 80 ℃ for protein expression and immunostaining detection.
5) The Western-blot method is used for detecting the expression of proteins such as Camk2α, camk2α -K42 and the like in the brain tissue of the mouse.
6) ELASA chemical titration was used to quantify the acetylation of CaMK 2. Alpha. In the mouse brain. First, purified His-K was used42 Ac Mixing of CaMK2 alpha and normal His-CaMK2 alpha protein at different percentages (0.5%, 1%,2%,4%, 8%), binding of protein by CaMK2 alpha antibody in 96-well plates, followed by Anti-K42 Ac Camk2α detection, on-machine reading OD450 values, plotting the acetylation ratio and the chemical standard curve y=0.056 x of OD 450. Because camk2α is one of the proteins with the largest brain content, about one percent of the proteins in the whole cerebral cortex, the total protein amount of each hole can be calculated relatively easily, after the consistent loading amount of each hole protein is ensured, the OD450 values of samples at different times are compared with a standard curve, the chemical quantitative curves (n=6) of the acetylation modification of the K42 site of the camk2α at different time points are obtained according to the standard curve, and the corresponding percentage value of the acetylated camk2α at each time point is calculated through chemical quantitative experiments.
(2) In vitro experiments
1. Behavioural experiments: autonomous movement, forelimb extension movement, screen experiments, haptic response, tail suspension experiments, and the like. Before each behavioural experiment starts, in order to relieve anxiety emotion of C57BL/6 mice (clean male wild type mice fed with water and aspirin or ethyl acetylsalicylate), an experiment operator can touch and stroker the C57BL/6 mice before the experiment, so that the mice are slowly adapted to the touch of the experiment operator, and the tension anxiety emotion of the mice is relieved as much as possible.
1) The autonomous movement specifically comprises the following steps: (1) during experiments, C57BL/6 mice are sequentially placed into a new animal cage box (length 27 cm. Times.width 27 cm. Times.height 38 cm); (2) recording activity conditions of the C57BL/6 mice in 5min by using Anymaze tracking recording software, and recording the wall collision times of the mice to score according to the standard; (3) after the completion, the C57BL/6 mice are taken out and put back into the original rearing cage box, urine and feces left by the C57BL/6 mice in the activity detection box are cleaned, the smell of the C57BL/6 mice is removed by 10% alcohol, and then the next C57BL/6 mice are put into the detection box.
2) The specific experimental steps of the stretching movement of the forelimbs include: (1) taking out a C57BL/6 mouse from the rearing cage box, placing the mouse on a whiteboard (with enough large area), and lifting the tail of the mouse by hands to ensure that only the forelimbs of the mouse can touch the whiteboard, so that the mouse naturally crawls; (2) recording forelimb extension conditions of the C57BL/6 mice when forelimbs climb on a whiteboard by using Anymaze tracking recording software, and scoring according to the standard; (3) after the completion, the C57BL/6 mice are taken out and put back into the original rearing cage box, urine and feces remained by the C57BL/6 mice in the activity detection box are cleaned, the smell of the C57BL/6 mice is removed by 10% alcohol, and then the next C57BL/6 mice are put on a white board.
3) The screen experiment comprises the following specific experimental steps: (1) preparing an iron screen (with a large enough area) inclined at 45 degrees; (2) taking out a C57BL/6 mouse from the rearing cage box, and placing the mouse in the center of the inclined screen; (3) recording the motion condition and climbing condition of the C57BL/6 mouse on a screen by using Anymaze tracking recording software, and scoring according to the standard; (4) after the completion, the C57BL/6 mice are taken out and put back into the original rearing cage box, urine and feces left by the C57BL/6 mice in the activity detection box are cleaned, the smell of the C57BL/6 mice is removed by 10% alcohol, and then the next C57BL/6 mice are put on a screen.
4) The haptic response was specifically experimental: (1) in the experiment, C57BL/6 mice are sequentially placed into a new animal cage box (length 27cm is multiplied by width 27cm is multiplied by height 38 cm), two beards and two bodies of the mice are lightly touched by cotton swabs respectively, and whether the responses of the mice are different or not is observed; (2) recording the difference conditions of the two side reactions of the C57BL/6 mice by using Anymaze tracking recording software and scoring according to the standard; (3) after the completion, the C57BL/6 mice are taken out and put back into the original rearing cage box, urine and feces left by the C57BL/6 mice in the activity detection box are cleaned, the smell of the C57BL/6 mice is removed by 10% alcohol, and then the next C57BL/6 mice are put into the detection box.
5) The specific experimental steps of the tail suspension experiment include: (1) taking out a C57BL/6 mouse from the rearing cage box, lifting the tail of the mouse by hands to suspend the mouse, taking a whiteboard (with a large enough area) as a background to enable tracking and recording to be clear, and observing the body symmetry of the movement of the limbs of the mouse; (2) recording limb movement conditions of the C57BL/6 mice in tail suspension by using Anymaze tracking recording software, and scoring according to the body state symmetry by using the standard; (3) the C57BL/6 mice are put back into the original rearing cage box after 3-7 seconds of tail suspension, and the next mouse is tested after the smell of the C57BL/6 mice is removed by 10% alcohol. Treatment of carcasses: the left brain blocks after trimming and animal carcasses are put into a freezer with animal carcasses specified by brain functions for unified treatment.
2. Preparation of an acetylated modified rabbit polyclonal antibody specifically recognizing camk2α:
1) Aiming at the acetylation modification of the K42 site specificity of CaMK2 alpha, the invention selects No. 37-48 amino acid synthesis modified polypeptide (C-QEYYAK Ac IINTKK), after coupling to KLH, immunizing into rabbits, sacrificing the rabbits, purifying antibodies by serum, synthesizing a control peptide (C-QEYYAKIINTKK), and performing purification and enzyme-linked immunosorbent assay (ELISA) detection;
2) Protein (His-K42) modified by site-directed acetylation of whole K42 site is directly synthesized and purified in vitro by utilizing non-natural coding acetylated lysine technology Ac CaMK2α) and K258 site-directed acetylation modified protein (His-K258) Ac -CaMK2α)；
3) The purity of the protein was detected by coomassie brilliant blue staining, and then the specificity of the antibody in vitro was detected by using the purified site-directed acetylated protein, by constantly optimizing conditions and purifying the antibody a number of times;
4) Mice that passed gene knockout and point mutation demonstrated antibody specificity.
3. Construction of eukaryotic cell systems to express site-directed acetylated CaMK2 alpha protein:
1) First, a single plasmid vector capable of expressing orthogonal pairs of aarsts and trnnacua simultaneously was constructed and optimized: the aaRSs/trnacoa orthogonal pair selected is the actkr 3/trnacoa orthogonal pair, as it has been demonstrated to be able to efficiently read through the amber codon UAG in mammalian cell lines, mouse forebrain, integrating the unnatural amino acid acetyl-lysine into the protein of interest;
2) After the tRNACUA was placed on the U6 promoter, four copies of the U6-tRNACUA expression module were constructed. To increase expression of tRNACUA, it is typically placed downstream of the U6 promoter, which initiates transcription of tRNACUA, and additionally increasing the copy number of tRNACUA can also increase the coding efficiency of the gene.
3) In order to allow efficient expression of AcKR3 in the brain of mice, the EF1a promoter was selected to initiate transcription and expression of AcKR 3. Experiments prove that EF1a is a eukaryotic strong promoter and can stably exist in mammalian cells and is not easy to silence.
4) Cloning of four copies of the U6-tRNACUA expression cassette onto the AcKR3 expression plasmid successfully constructed four copies of the U6-tRNACUA and the single plasmid expression system for AcKR3 expression (RS (tRNACUA) 4X ). To determine and verify RS (tRNACUA) 4X Simultaneously introducing an amber codon (amb) at the 39 th position of the EGFP gene to construct an EGFP amb single fluorescence reporter molecule;
5) EGFPamb reporter plasmid and four copies of RS of tRNACUA (tRNACUA) 4X The expression plasmids are respectively transfected into HEK293T cells together, and the cells are respectively cultured by a culture medium containing and not containing 1mM of acetyl-lysine, if the 39-amb codon of EGFPIMb is read through, the full-length EGFP can be expressed, green fluorescence can be seen under a fluorescence microscope, and the acetyl-lysine is integrated into EGFP fluorescent proteins;
6) After 24h of cell transfection, expression of green fluorescent protein was observed only in cells cultured in 1mM acetyl-lysine medium, indicating that acetyl-lysine has been integrated into EGFP protein;
7) Detection RS (tRNA) CUA ) 4X Expression efficiency of plasmids in eukaryotic cells.
(3) And (3) statistics: study results data were statistically analyzed using Prism 5.0 software and experimental data results were expressed as mean ± standard error (mean ± SEM). Two-group statistics were performed with two-tailed unpaired t-test (two-tailed unpaired t-tests) Student's t-test, and one-way anova was used for the multi-group statistics. p <0.05 is statistically different.
(4) Procedural conclusion:
1) According to the mass spectrum result, the acetylation modification level of most proteins in the brain of the mice is increased after 14 days of feeding aspirin, wherein the lysine 42 site and 258 site of Camk2α are most prominent, as shown in FIG. 1;
2) Under in vitro conditions, aspirin can directly improve the acetylation level of Camk2α, has a dose-dependent characteristic, and is detected by LC/MS mass spectrometry, K42 and K258 are found to be main acetylation sites, as shown in FIG. 1 and FIG. 2;
3) Quantification of acetylated camk2α using ELASA chemotitration format found that aspirin administration increased the camk42 site acetylation level of camk2α in a time dependent manner and increased about six-fold after stabilization (the level of acetylation modification at the K42 site of camk2α increased from about 0.52% of normal and stabilized at 3.01%) as shown in fig. 3;
4) The cell subfraction fractionation technology is adopted to find that the K42 site acetylation modification of CaMK2 alpha is mainly enriched in cytoplasm, and the proportion of acetylation modification in synapses is low (the proportion of acetylation in cytoplasm reaches 13 percent, and the proportion of the K42 site acetylation modification of CaMK2 alpha in synapses is low and is only 2 percent) as shown in figure 2;
5) The K42 site acetylation modification did not affect CaMK 2. Alpha. And Ca 2+ The binding between/CaM is shown in fig. 4;
6) The K42 site is an ATP binding site of calmodulin kinase, and two methods of calculation structural simulation and ATP immunoprecipitation are adopted to find that the acetylation modification of the K42 site significantly damages the combination of CaMK2 alpha protein kinase and ATP, as shown in FIG. 4;
7) In vitro autophosphorylation experiments show that the acetylation of the K42 site damages autophosphorylation of the T286 site of CaMK2 alpha, but the acetylation of the K258 site has no effect on the autophosphorylation of CaMK2 alpha;
8) By adopting a kinase kit to carry out a substrate phosphorylation experiment, the acetylation of the K42 site of CaMK2 alpha can inhibit the phosphorylation of the CaMK2 alpha to a substrate, but the acetylation of the K258 site does not influence the phosphorylation function of the CaMK2 alpha to the substrate, and the ratio of 13% of the acetylation modification of the K42 site can obviously inhibit the overactivation of the CaMK2 alpha per se, and the CaMK2 alpha is subjected to Ca 2+ CaM/ATP and Ca2+/CaM/H 2 O 2 Overactivation under both conditions had significant inhibitory effects, as shown in figure 4;
9) Kinase experiments have found that aspirin is not a direct inhibitor of camk2α and inhibits its kinase activity, as shown in fig. 5;
10 Interference with acetylation of CaMK2 alpha protein K42 site impairing aspirin protective effect on ischemic stroke mice model as shown in fig. 5;
11 Through construction of a cerebral apoplexy model mouse, injection of an inhibitory peptide ventricle and a cognition behavioural experiment by a Carcia score method, analysis and statistics are carried out from three layers of mortality, cognitive function score and infarct size, and after aspirin is found to have better neuroprotection effect on the cerebral apoplexy model mouse, injection of the inhibitory peptide reduces acetylation modification of K42 site, the neuroprotection effect of aspirin on the three layers is weakened, and the acetylation modification of the CaMK2 alpha protein K42 site is proved to be necessary for the neuroprotection effect of aspirin, as shown in figure 5;
12 By constructing eukaryotic cell system to express site-directed acetylation CaMK2 alpha protein, it is found that the CaMK2 alpha protein with site-directed acetylation modification of K42 site expressed in forebrain of mice can play a direct neuroprotective role in cerebral stroke models of mice, that is, the acetylation modification of the K42 site of the CaMK2 alpha protein can fully play a neuroprotective role, as shown in FIG. 6.
13 Ethyl acetylsalicylate as a modification of aspirin can reduce anticoagulation and exert neuroprotective effects, as shown in fig. 8:
14 Testing and comparing the clotting time of mice 14 days after feeding aspirin and ethyl acetylsalicylate by clotting experiments, it was found that aspirin increased the total clotting time, while ethyl acetylsalicylate significantly decreased clotting time, as shown in fig. 8;
15 Acetylsalicylic acid ethyl ester as an improvement of aspirin, which maintains the ability to acetylate the CaMK2 alpha protein K42 site, and which can better increase the acetylation level of CaMK2 alpha than aspirin, as shown in FIG. 8;
16 Ethyl acetylsalicylate reduced mortality 24h after stroke in mice, reduced cognitive impairment, and reduced infarct size compared to control group, had neuroprotection, and was not significantly different from aspirin positive, as shown in figure 8.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Claims (15)

1. A biomarker, wherein the biomarker is an acetylation site of calmodulin-dependent protein kinase 2 a (CaMK 2 a) within a neural cell; the amino acid sequence of CaMK2 alpha is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2; the acetylation sites are K42 and K286.
2. Use of the biomarker according to claim 1, in the manufacture of a medicament for the prevention and/or treatment of stroke diseases, cerebral ischaemic diseases, characterised in that the medicament is aspirin and/or an aspirin derivative, modification.
3. The use according to claim 2, wherein the medicament comprises the aspirin derivative ethyl acetylsalicylate.
4. The use according to claim 2, wherein the aspirin and/or aspirin derivatives, modifications, alterations increase the level of acetylation of camk2α by acetylating intracellular calcium ion-calmodulin dependent protein kinase 2α (camk2α), inhibiting the kinase activity of camk2α; the acetylization sites of aspirin and/or aspirin derivatives, modifications and remodels on CaMK2 alpha comprise K42 and K286.
5. The use of claim 2, wherein the acetylation modification of the K42 site of CaMK2 alpha is enriched in cytoplasm; acetylation of the K42 site of CaMK 2. Alpha. Inhibits its autophosphorylation and phosphorylation to a substrate, but does not affect CaMK 2. Alpha. And Ca 2+ between/CaMAnd (5) combining.
6. The use according to claim 2, characterized in that the use comprises the use of aspirin and/or aspirin derivatives, modifications, alterations in the preparation of a medicament for the prevention and/or treatment of cerebral stroke diseases, recurrent cerebral ischemia, in the preparation of a medicament for patients who cannot use/cannot use a large dose of aspirin but who are at risk of cerebral ischemia at the same time, or in the preparation of a medicament for the replacement therapy of aspirin.
7. A modulator of a biomarker, comprising an agonist, inhibitor or antagonist of the biomarker of claim 1.
8. A pharmaceutical/pharmaceutical composition comprising a biomarker according to claim 1, or a compound that can also bind to or react with a biomarker according to claim 1.
9. The drug/pharmaceutical composition according to claim 8, characterized in that it comprises aspirin and/or aspirin derivatives, modifications, modified substances.
10. A detection reagent/kit comprising an active ingredient useful for detecting the biomarker of claim 1 and its level of acetylation.
11. A polyclonal antibody for specifically recognizing the acetylated CaMK2 alpha at K42 site features that the modified polypeptide (C-QEYYAK) is prepared from the amino acids 37-48 Ac IINTKK) coupled with KLH, and obtaining the strain after immunization of rabbits; the antibody can specifically recognize the CaMK2 alpha protein acetylated at the K42 site, can not recognize the wild non-acetylated CaMK2 alpha protein and can not recognize the CaMK2 alpha protein acetylated at other sites such as K258.
12. Use of a modulator of a biomarker according to claim 7, a drug/pharmaceutical composition according to claim 8, a detection reagent/kit according to claim 10, or an antibody according to claim 11 for the prediction, assessment, prevention and/or treatment of stroke diseases, cerebral ischemic diseases, recurrent cerebral ischemic diseases, for the preparation of a medicament for patients who cannot use/cannot use a large dose of aspirin but are at risk of cerebral ischemia at the same time, or for the preparation of a medicament for substitution therapy of aspirin.
13. The use according to claim 12, wherein the modulator, drug/pharmaceutical composition, detection reagent/kit may be used alone or in combination with other drugs.
14. A method of screening for a candidate drug for preventing and/or treating a stroke disease, a cerebral ischemia disease, the method comprising detecting an effect of the candidate drug on a biological effect of the biomarker of claim 1 in a subject or a sample obtained from a subject, comprising detecting a level of acetylation of the K42 site of camk2α or a kinase activity of camk2α in the subject or the sample obtained from the subject.
15. A method of preventing and/or treating stroke disease, cerebral ischemia disease in a subject in need thereof, or a method of predicting and/or prognostic evaluation of whether or not the patient is suitable for aspirin and/or aspirin derivatives, modifications, alterations to prevent and/or treat stroke disease, cerebral ischemia disease, comprising detecting the level of acetylation at the K42 site of camk2α or the kinase activity of camk2α of the biomarker of claim 1.
CN202210840260.4A 2022-07-18 2022-07-18 Marker for preventing and protecting cerebral ischemia diseases and application of aspirin derivative in preventing and protecting cerebral ischemia diseases Pending CN117417914A (en)

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FI20105715A0 (en) * 2010-06-18 2010-06-18 Helsingin Yliopisto A polyclonal antibody that binds to acetylated HMGB1
BR112014024537A2 (en) * 2012-04-02 2017-08-08 Berg Llc methods to identify modulators of a biological system, a disease process, and angiogenesis
CN103900893A (en) * 2013-05-20 2014-07-02 上海华盈生物医药科技有限公司 Kit for gathering protein modified by acetylization, as well as method and application of kit
US20200057075A1 (en) * 2017-02-20 2020-02-20 The Regents Of The University Of California Serologic assay for silent brain ischemia
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