CN103566362A - Application of recombinant ADAMTS13 to preparation of cerebral hemorrhage medicaments - Google Patents
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Abstract
The invention belongs to the field of biological pharmacy, relates to application of recombinant ADAMTS13 to preparation of cerebral hemorrhage medicaments, and especially relates to application of recombinant ADAMTS13 to preparation for medicaments for reducing cerebral hemorrhage caused by tPA thrombolytic therapy on cerebral ischemia. Experiment results show that: VEGF generated by tPA induction at ischemic condition can be blocked by a specific inhibitor of RhoA or Akt, and recombinant ADAMTS13 substantially reduces activation of RhoA and phosphorylation of Akt, which means that recombinant ADAMTS1 is capable of inhibiting tPA-induced VEGF expression through RhoA and Akt pathways. The recombinant ADAMTS13 provided by the invention helps to mitigate damage of tPA to blood cerebral barrier and further to reduce cerebral hemorrhage caused thereby by inhibiting cerebrovascular permeability increase mediated by RhoA and Akt, and therefore combination usage of recombinant ADAMTS13 and tPA is a novel countermeasure and means for increasing thrombolysis security.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to the purposes of restructuring ADAMTS13 in preparing cerebral hemorrhage medicine, the purposes of especially recombinating in the ADAMTS13 cerebral hemorrhage medicine that tPA thromboembolism treatment causes after preparation reduces cerebral ischemia.
Background technology
Research shows, apoplexy is to cause one of disabled and main causes of death of the mankind; And the tPA(Tissue Plasminogen Activator of tissue-type plasminogen activator) because of it, there is powerful thrombolytic effect, become the most effective medicine of current treatment apoplexy; But use tPA thrombolytic to have the risk that increases cerebral hemorrhage, therefore, how to overcome the cerebral hemorrhage that tPA causes, to reducing disabling of apoplexy, have great importance with fatality rate.
Described tPA is a kind of serine protease, and it plays an important role in maintaining blood coagulation and fibrinolysis process; TPA not only plays regulatory role normal central nervous system's physiology and neuron plasticity, also plays a part crucial aspect central nervous system pathology.Described tPA promotes neuronal death, the increase ischemic brain injury that excitotoxin causes, gene knockout tPA or medicine suppress tPA ischemia brain is all played to significant protective effect.In addition, tPA has been proved to be can be by promoting the activation of Matrix Metalloproteinase-9 (MMP-9) and transcription factor NF-KB and the degraded that promotes Akt phosphorylation and extracellular matrix, and aggravation lacks breaking of hemoendothelial damage and blood brain barrier.Existing a large amount of experiment and clinical evidences show, tPA causes that breaking of blood brain barrier is that it causes one of immediate cause of cerebral hemorrhage; Therefore, the blood brain barrier that alleviates tPA mediation breaks, and will effectively reduce the cerebral hemorrhage that tPA thrombolytic causes.
Studies show that, vWF ELISA von Willebrand factor (VWF) is the glycoconjugates albumen that endotheliocyte and megalokaryocyte produce; In addition, ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) is newfound a kind of metalloproteases being present in blood plasma in recent years, the most important function of described ADAMTS13 is that the super large VWF of newly secretion is cracked into the coreceptor form that less adhesiveness is low, thereby reduces the formation of thrombosis; Described VWF is ADAMTS13 known unique cracking substrate at present, and central nervous system, VWF expressed in abundance in endotheliocyte,, but knows little about it to VWF role in regulating blood-brain barrier permeability.There are some researches show, at cerebral infarction, cerebral hemorrhage and subarachnoid hemorrhage, these break in relevant disease with blood brain barrier, and the VWF in blood plasma expresses obviously and raises; In addition, suffer from the patient of cardiovascular disease after carrying out thromboembolism treatment, the VWF in blood plasma also obviously raises.Studies have found that recently, VWF gene delection shields to cerebral ischemia mice, and in contrast, ADAMTS13 gene knockout causes cerebral ischemia focus obviously to increase, and restructuring ADAMTS13 significantly alleviates Acute cerebral ischemia.
Up to now, there is not yet relevant restructuring ADAMTS13 for reducing the report of the cerebral hemorrhage that tPA thromboembolism treatment causes after cerebral ischemia.
The list of references relevant with the present invention has:
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Summary of the invention
The object of this invention is to provide the purposes of restructuring ADAMTS13 in preparing cerebral hemorrhage medicine, the purposes of especially recombinating in the ADAMTS13 cerebral hemorrhage medicine that tPA thromboembolism treatment causes after preparation reduces cerebral ischemia.
The present invention confirms that through experiment intracerebral ventricle injection VWF causes the quick increase of non-ischemia blood-brain barrier of mice permeability, and after also confirming VWF lyases ADAMTS13 cerebral ischemia capable of blocking tPA to cerebrovascular destruction, thereby reduce the cerebral hemorrhage that tPA thrombolytic brings out.
Particularly, the present invention has carried out the experiment comprising the following steps:
(1) intracerebral ventricle injection and cerebrovascular permeability are measured;
(2) animal brain ischemia model;
(3) behavior determination;
(4) measurement of cerebral hemorrhage;
(5) Western Blot experiment;
(6) SABC;
(7) data analysis.
Result demonstration,
(1) restructuring ADAMTS13 blocks the increase of the blood-brain barrier permeability that in non-cerebral ischemia-reperfusion, tPA injection causes
First confirm that normal mouse intracerebroventricular injection tPA can cause the increase of blood-brain barrier permeability; Then use non-ischemia mice, by intracerebroventricular VWF, intravenous injection Evans blue then, after 1 hour, perfusion is got brain, the seepage discharge of Evans blue in detection by quantitative brain, result shows, is similar to injection tPA, VWF significantly increases the permeability of blood brain barrier; Yet tPA and VWF joint injection cause that degree and both independent uses that blood-brain barrier permeability increases do not have notable difference, show that tPA and VWF affect the permeability of blood brain barrier by same path; Further, result demonstration, tricorn gives 50ng or 100ng restructuring ADAMTS13 can significantly reduce the Evans blue seepage discharge in the mouse brain of injecting tPA, and inject separately the ADAMTS13 that recombinates, the permeability of blood brain barrier is not affected;
(2) destruction of tPA to blood brain barrier after restructuring ADAMTS13 inhibition cerebral ischemia
Adopt 45 minutes focal cerebral ischemia models of mice, detected ZO-1(main tight junction protein) and the variation expressed of collagen IV (a kind of main basement membrane of blood vessel albumen), result shows, cerebral ischemia obviously reduces the expression of ZO-1, suitably reduces the expression of collagen IV; Compare with PBS processed group, two hours posterior veins of cerebral ischemia are injected the obvious minimizing that tPA causes ZO-1 and collagen IV; Yet 3 hours tricorns ADAMTS13 that recombinates significantly alleviates ZO-1 that tPA causes and the degraded of collagene IV after cerebral ischemia; Further, with matched group comparison, tPA significantly increases the seepage discharge of cerebral ischemia-reperfusion hemisphere Evans blue; Result shows, injection restructuring ADAMTS13 reverses the change of the blood-brain barrier of mice permeability that tPA causes completely, alone restructuring ADAMTS13 on cerebral ischemia after the not impact of permeability of blood brain barrier;
(3) impact of restructuring ADAMTS13 on cerebral hemorrhage and function of nervous system
By spectrophotometric quantitative measurement, result shows, within after cerebral ischemia 2 hours, injects tPA and causes the cerebral hemorrhage after 24 hours obviously to increase; After cerebral ischemia, 3 hours Intraventriculars ADAMTS13 that recombinates suppresses the cerebral hemorrhage of tPA induction; The ADAMTS13 that recombinates separately does not have a significant effect to cerebral hemorrhage;
In addition, function of nervous system marks and shows, compare with the mice of PBS processed group, tPA processed group mice shows the decline of serious neurological deficit and motor function; And function of nervous system's scoring of restructuring ADAMTS13+tPA group mice and motor function are significantly better than the mice that gives separately tPA;
(4) expression of VEGF in the vascular endothelial cell that restructuring ADAMTS13 reduction tPA causes
Adopt Western Blot experiment, result shows, compares with sham operated rats, and it is (consistent with former report that cerebral ischemia significantly increases the expression of VEGF
34); Compare with the cerebral ischemia mice that gives PBS, giving the expression that tPA causes VEGF further increases;
Immunohistochemical staining result shows, gives tPA and cause that the expression of VEGF in the vascular endothelial cell of infarction surrounding zone significantly increases; Although also can be observed the expression of VEGF in astrocyte and neuron, the impact that not processed by tPA;
The result of Western Blot experiment shows, restructuring ADAMTS13 significantly suppresses the increase of the vegf expression that tPA causes, consistent with this result, in the vascular endothelial cell of infarction surrounding zone, the immunocompetence of the VEGF of tPA induction also significantly reduces;
The expression of Ang-1 and Ang-2 in mensuration cerebral tissue, result demonstration, the expression of Ang-1 and Ang-2 is basically identical between reason group throughout, shows that Ang-1 and Ang-2 do not participate in regulating the cerebral hemorrhage of tPA induction;
(5) vegf expression of restructuring ADAMTS13 inhibition tPA induction is relevant with Akt path with RhoA
Mice is divided into cerebral ischemia group and tPA processed group, the inhibitor fasudil of intravenous injection RhoA and the inhibitor wortmannin of Akt, result demonstration, fasudil and wortmannin all can suppress the rise of the vegf expression of tPA induction;
Western Blot experiment result shows, cerebral ischemia obviously increases the activity of RhoA, and induces the phosphorylation of Akt; After giving tPA, above-mentioned effect is further expanded; On the contrary, restructuring ADAMTS13 significantly suppresses the RhoA activation of tPA induction and the phosphorylation of Akt; The expression of total Akt does not detect difference between each group;
The above results shows, restructuring ADAMTS13 plays by suppressing the activation of RhoA and the phosphorylation of Akt the effect of disturbing tPA-VEGF path.
Experimental result of the present invention shows, the VEGF that tPA induction produces under ischemia condition can be blocked by the specific inhibitor of RhoA or Akt, and restructuring ADAMTS13 significantly reduces the activation of RhoA and the phosphorylation of Akt, the ADAMTS13 that shows to recombinate realizes by RhoA and Akt path the inhibition of the vegf expression of tPA induction.
Restructuring ADAMTS13 of the present invention increases by suppressing the cerebrovascular permeability of RhoA and Akt mediation, alleviate tPA thereby the infringement of blood brain barrier is reduced to the cerebral hemorrhage that it causes, therefore, combining use restructuring ADAMTS13 and tPA is a kind of novel countermeasure and the means that increase thrombolytic safety.
Accompanying drawing explanation
Fig. 1 has shown under non-ischemia condition, the increase of the blood-brain barrier permeability that restructuring ADAMTS13 inhibition tPA causes, wherein,
A: intracerebroventricular injection PBS, tPA, VWF or tPA add after VWF, the measured value of Evans blue seepage discharge in 1 hour hindbrain of intravenous injection Evans blue;
B: intravenous injection PBS, tPA, tPA and 50 or 100ng restructuring ADAMTS13 joint injection, restructuring ADAMTS13 is separately after injection, the measured value of Evans blue seepage discharge in 1 hour hindbrain of intravenous injection Evans blue;
Numerical value adopts mean+standard deviation, every group of n=7-8, * P<0.05.
Fig. 2 has shown that restructuring ADAMTS13 suppresses the blood brain barrier of tPA induction after cerebral ischemia and breaks, wherein,
A, C: cerebral ischemia 24 hours, sham operated rats and matched group, tPA administration group, tPA+ restructuring ADAMTS13 administration group, the representative immunoblotting photo of zo-1 and collagen IV;
B, D: the quantitative assay of the ZO-1 of each processed group and collagen IV; Value adopts mean+standard deviation, sham operated rats n=4, and other respectively organizes n=6, * P<0.05;
E: cerebral ischemia 24 hours, matched group, tPA administration group, the representative diagram that the Evans blue that tPA+ restructuring ADAMTS13 administration group and restructuring ADAMTS13 are individually dosed group oozes out;
F: the quantitative analysis that the Evans blue of each processed group oozes out; Value adopts mean+standard deviation, every group of n=7, * P<0.05.
Fig. 3 has shown the impact of function of nervous system after restructuring ADAMTS13 is on cerebral hemorrhage and cerebral ischemia, wherein,
A: representational brain dorsal surface and crown section show the cerebral ischemia cerebral hemorrhage situation of 24 hours, and picture comes from respectively matched group, tPA administration group, the mice that tPA+ restructuring ADAMTS13 administration group and restructuring ADAMTS13 are individually dosed group; Arrow refers to the large-area hemorrhage of the mouse brain ischemic region that gives tPA;
B: the result with spectrophotometer to cerebral hemorrhage detection by quantitative; Value adopts mean+standard deviation, every group of n=8, * P<0.05.(C) function of nervous system scoring and (D) cerebral ischemia 24 hours, matched group, tPA administration group, the motor behavior of tPA+ restructuring ADAMTS13 administration group and individually dosed group of mice of restructuring ADAMTS13; Value adopts mean+standard deviation, every group of n=8, * P<0.05.
Fig. 4 has shown that restructuring ADAMTS13 reduces the vegf expression in the vascular endothelial cell of tPA induction, wherein,
A: cerebral ischemia 24 hours, sham operated rats mice, matched group, tPA administration group, the representative immunoblotting picture of the vegf expression that tPA+ restructuring ADAMTS13 administration group and restructuring ADAMTS13 are individually dosed group;
B: the quantitative assay result of the vegf expression of each processed group; Value adopts mean+standard deviation, sham operated rats n=4, and other respectively organizes n=6, * P<0.05;
C: cerebral ischemia 24 hours, gives tPA group or tPA+ restructuring ADAMTS13 group mice infarction surrounding zone VEGF(dark) and copolymerization Jiao picture of endotheliocyte (CD31, bright) SABC; Asterisk indication lumen of vessels; Scale: 15 μ m;
D, E: cerebral ischemia 24 hours, sham operated rats mice, matched group, tPA administration group, the representative immunoblotting picture that tPA+ restructuring ADAMTS13 administration group Ang-1 and Ang-2 express;
F, G: each organizes the quantitative assay result of Ang-1 and Ang-2 expression; Value adopts mean+standard deviation, and each organizes n=6, * P<0.05.
Fig. 5 has shown that the vegf expression of restructuring ADAMTS13 inhibition tPA induction is relevant with Akt path with RhoA, wherein,
A, C: cerebral ischemia 24 hours, gives tPA group, the representative immunoblotting picture of vegf expression in the mice ischemia side brain of tPA+ wortmannin or fasudil group;
B, D: each organizes the quantitative assay result of vegf expression; Value adopts mean+standard deviation, every group of group n=5, * P<0.05;
E, F: cerebral ischemia 24 hours, sham operated rats mice, matched group, tPA administration group, tPA+ restructuring ADAMTS13 administration group phosphorylation Akt, total Akt and the representational immunoblotting picture of RhoA;
G, H: each organizes the quantitative assay result of phosphorylation Akt and RhoA; Value adopts mean+standard deviation, sham operated rats n=4, and other respectively organizes n=6, * P<0.05.
Specific embodiment
1, materials and methods
(1) reagent and antibody
Evans blue, Drabkin, paraformaldehyde, D-Glucose, Methanamide, wortmannin (buying in Sigma-Aldrich) (St Louis, the MO， U.S.), people's Recomposed tPA (Ai Tongli) is purchased from Boehringer Ingelheim (Mannheim, Germany), people VWF albumen is bought from Haematologic Tecnologies (the Essex Junction ，VT， U.S.), people recombinates ADAMTS13 purchase from R & D systems (neapolis, the MN， U.S.), fasudil is bought from Calbiochem (San Diego, the CA， U.S.).Primary antibodie comprises rabbit anti-zonula occludens (ZO-1) (Invitrogen, Camarillo, CA, the U.S.), rabbit anti-collagen IV (Santa Cruz Biotechnology, Santa Cruz, CA, the U.S.), rabbit anti-VEGF (VEGF) (Abcam, MA, the U.S.), goat anti-Angiopoietin (Ang)-1 (Santa CruzBiotechnology, Santa Cruz, CA, the U.S.), goat anti-Ang-2 (Santa Cruz Biotechnology, Santa Cruz, CA, the U.S.), rabbit anti-phospho-Akt (serine 473) (Cell Signaling Tecnology, Beverly, MA, the U.S.), rabbit anti-Akt (Cell Signaling Technology, Beverly, MA, the U.S.), rabbit anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, CA, the U.S.) rat anti-CD31 (PECAM-1, BD Pargen, San Diego, CA, the U.S.), rabbit anti-β-actin (Cell Signaling technology, Beverly, MA, the U.S.). two anti-comprising: Alexa Fluor 594donkey anti-rabbit immunoglobulin G (IgG), Alexa Fluor 488 donkey anti-rat IgG (Invitrogen, Camarillo, CA, the U.S.).
(2) intracerebral ventricle injection and cerebrovascular permeability are measured
All experiments are through shanghai Medicine institute of Fudan University animal love and use committee to check and approve.The present invention adopts male adult C57 mice (Shanghai Slac Experimental Animal Co., Ltd. provides, Shanghai, China).
Mice is placed in to stereotaxic instrument (Narisige Scientific Instrument Laboratory, Tokyo, Japan), with the mist continuous narcosis containing 1-1.5% isoflurane, 30% oxygen, at following coordinate: 0.2mm after bregma, 1.0mm is opened on side, veutro degree of depth 3mm, intracranial is injected respectively 3 μ l PBS, tPA(585ng), VWF (540ng), tPA and VWF, tPA and restructuring ADAMTS13 (50 or 100ng) or restructuring ADAMTS13 (100ng), be 15 minutes inject time; Then, intravenous injection Evans blue dye (4%, be dissolved in PBS, 4ml/kg), after 60 minutes, mice is through cardiac perfusion PBS, and cerebral tissue is placed in formamide solution and soaks 72 hours; Adopt spectrophotometer (Thermo BioMate 3S, Thermo Scientific, the U.S.) at 620nm wavelength place, to measure the volumes of extravasation result of Evans blue in cerebral tissue.
(3) animal brain ischemia model
Cerebral ischemic model (MCAO) is made according to the method for prior art
25,27, male mice, with after the mist anesthesia containing 1-1.5% isoflurane, 30% oxygen, 70% nitrogen, inserts through 7.0 nylon of silica gel parcel end and fastens line blocking-up middle cerebral artery through right side internal carotid artery; 45 minutes backed off after randoms of ischemia are fastened line and are implemented to pour into again; Use hot plate (World Precision Instruments, the Sarasota ，FL， U.S.) to maintain mouse temperature at 37 ± 0.5 ℃; By laser Doppler flowmetry (Perimed, Stockholm, Sweden), confirm the thromboembolism of middle cerebral artery and pour into again situation; For increasing cerebral hemorrhage amount, cerebral ischemia gives mouse peritoneal injection 50%D-glucose (6ml/kg) after 15 minutes
28,29; After cerebral ischemia 2 hours, with micro-sampling pump (World Precision Instruments, the Sarasota ，FL， U.S.) vein, inject 1mg/kg(bolus) tPA (10mg/kg) or PBS, remaining 9mg/kg slowly injected through 30 minutes; Restructuring ADAMTS13 (100ng is dissolved in 3 μ l PBS) or PBS give through right brain tricorn for 3 hours after cerebral ischemia; Wortmannin (15 μ g/kg are dissolved in 100 μ l containing in the normal saline of 1% dimethyl sulfoxide [DMSO]) or containing the normal saline of 1% dimethyl sulfoxide before cerebral ischemia through intravenous injection; Fasudil (10mg/kg is dissolved in 100 μ l normal saline) or normal saline after cerebral ischemia 30 minutes by intravenous injection; After mouse brain ischemia 23 hours, by the Evans blue(2ml/kg of intravenous injection 2%); The Evans blue volumes of extravasation result of ischemia side brain hemisphere is measured by the method for narration above.
(4) behavior determination
After cerebral ischemia 24 hours, adopt double-blind method to mark to mice nervous function damage:
0 minute, impassivity function damage;
1 minute, can not full extension left side fore paw;
2 minutes, the resistance of thrust is weakened;
3 minutes, turn-take left spontaneously;
4 minutes, autonomic activities disappearance or unconscious.
In motor behavior test, mice is put into test box (the Med Associates of 27.3 * 27.3 * 40cm, the U.S.), by infrared camera, automatically record the distance that mice was advanced in 30 minutes, adopt SOD-811 analysis software (Med Associates Inc., the St Alban's ，VT， U.S.) analytical data.
(5) measurement of cerebral hemorrhage
After ischemia 24 hours, give the excessive chloral hydrate of injected in mice, with PBS, carry out cardiac perfusion; Collect ischemia side cerebral tissue, add 500 μ l Drabkin solution, homogenate, 4 ℃ of 13000rpm centrifugal 30 minutes; Collect supernatant, use spectrophotometer (Thermo BioMate 3S, Thermo Scientific, the U.S.) in its optical density value of 540nm wavelength measurement; The blood production standard curve that adopts mice homology, calculates amount of bleeding corresponding to optical density value 25,30 by standard curve.
(6) Western Blot experiment
By the ischemia side cerebral tissue cerebral tissue corresponding with sham operated rats through lysate cracking; RIPA lysate (Millipore, Billerica, MA that cerebral tissue is being contained to protease inhibitor cocktails, the U.S.) in homogenate, centrifugal after, with BCA albuminometry (Thermo BioMate 3S, Thermo Scientific, the U.S.), detect protein concentration; The protein sample of equivalent after 8%, 10% or 12% Tris-glycine gels electrophoresis, is transferred on pvdf membrane respectively; Use the 5% skim milk room temperature of 1XTBST to seal 1 hour, 4 ℃ of overnight incubation of primary antibodie add two anti-incubated at room 1 hour after rinsing; Chemiluminescence agent (Millipore, the Billerica ，MA， U.S.) is measured system (Bio-Rad, the U.S.) with Bio-Image and is scanned and quantitative analysis after processing; Use β-actin as internal reference.
(7) SABC
After cerebral ischemia 24 hours, by mice with excessive chloral hydrate fiber crops deeply, then successively with the paraformaldehyde solution perfusion of PBS and 4%, get brain; Cerebral tissue dewaters through 30% sucrose; The PBS confining liquid sealing of the bovine serum albumin containing 1% and 5% normal donkey serum for the crown section of brain (20 μ m), by 4 ℃ of overnight incubation of antibody mixed liquor of the anti-VEGF of rabbit and large mouse-anti CD31; To cut into slices after rinsing, with the mixture of the Alexa Fluor anti-rabbit igg of 594 donkey and the Alexa Fluor 488Lv Mus Ig of the Chinese People's Anti-Japanese Military and Political College, hatch; Use Olympus FV1000 Laser Scanning Confocal Microscope to obtain image, FV10-ASW software carries out image processing.
(8) data analysis
Data represent with meansigma methods ± SD, adopt one factor analysis of variance relatively to carry out statistical analysis by many groups of Boneferroni; Adopt Kruskal – Wallis test to analyze by many groups of statistical analysiss that relatively carry out behavioristics's data of Dunn; Two groups relatively time, adopt the two tail Student t checks of pairing.P<0.05 has been considered as significant difference.
2, result
(1) restructuring ADAMTS13 blocks the increase of the blood-brain barrier permeability that in non-cerebral ischemia-reperfusion, tPA injection causes
First confirm that normal mouse intracerebroventricular injection tPA can cause the increase (as shown in Figure 1A) of blood-brain barrier permeability
28,31; In order to detect VWF, whether also can induce the increase of blood-brain barrier permeability, use non-ischemia mice, by intracerebroventricular VWF, intravenous injection Evans blue then, after 1 hour, perfusion is got brain, the seepage discharge of Evans blue in detection by quantitative brain; Result shows, is similar to injection tPA, and VWF significantly increases the permeability (as shown in Figure 1A) of blood brain barrier; Yet tPA and VWF joint injection cause that degree and both independent uses that blood-brain barrier permeability increases do not have notable difference, show that tPA and VWF affect the permeability of blood brain barrier by same path; Further can research VWF lyases ADAMTS13 suppress the caused blood-brain barrier disruption of tPA, result demonstration, tricorn gives 50ng or 100ng restructuring ADAMTS13 can significantly reduce the Evans blue seepage discharge (as shown in Figure 1B) in the mouse brain of injecting tPA; Because the restructuring ADAMTS13 of 100ng is more effective, in follow-up experiment, use the restructuring ADAMTS13 of 100ng; Injection restructuring ADAMTS13 is on the not impact of the permeability of blood brain barrier separately.
(2) destruction of tPA to blood brain barrier after restructuring ADAMTS13 inhibition cerebral ischemia
Adopt 45 minutes focal cerebral ischemia models of mice, detected ZO-1(main tight junction protein) and the variation expressed of collagen IV (a kind of main basement membrane of blood vessel albumen), result shows, cerebral ischemia obviously reduces the expression (as shown in Fig. 2 A and B) of ZO-1, suitably reduces the expression (as shown in Fig. 2 C and D) of collagen IV; Compare with PBS processed group, two hours posterior veins of cerebral ischemia are injected the obvious minimizing that tPA causes ZO-1 (as shown in Fig. 2 A and B) and collagen IV (as shown in Fig. 2 C and D); Yet 3 hours tricorns ADAMTS13 that recombinates significantly alleviates ZO-1 that tPA causes and the degraded of collagene IV after cerebral ischemia; Result also shows, with matched group comparison, tPA significantly increases the seepage discharge (as shown in Fig. 2 E and F) of cerebral ischemia-reperfusion hemisphere Evans blue, and injection restructuring ADAMTS13 reverses the change (as shown in Fig. 2 E and F) of the blood-brain barrier of mice permeability that tPA causes completely; And alone restructuring ADAMTS13 on cerebral ischemia after the not impact of permeability of blood brain barrier.
(3) impact of restructuring ADAMTS13 on cerebral hemorrhage and function of nervous system
By spectrophotometric quantitative measurement, result shows, within after cerebral ischemia 2 hours, inject tPA and cause the cerebral hemorrhage after 24 hours obviously to increase (as shown in Fig. 3 A and B), after cerebral ischemia, 3 hours Intraventriculars ADAMTS13 that recombinates suppresses the cerebral hemorrhage (as shown in Fig. 3 A and B) of tPA induction; The ADAMTS13 that recombinates separately does not have a significant effect to cerebral hemorrhage.
In addition, function of nervous system marks and shows, compare with the mice of PBS processed group, tPA processed group mice shows the decline of serious neurological deficit and motor function; Function of nervous system's scoring of restructuring ADAMTS13+tPA group mice and motor function are significantly better than the mice that gives separately tPA.
(4) expression of VEGF in the vascular endothelial cell that restructuring ADAMTS13 reduction tPA causes
The result demonstration of Western Blot experiment, compares with sham operated rats, and cerebral ischemia significantly increases the expression of VEGF, and this result is consistent with former report
34; Compare with the cerebral ischemia mice that gives PBS, giving the expression that tPA causes VEGF further increases (as shown in Fig. 4 A and B);
Immunohistochemical staining result shows, gives tPA and cause that the expression of VEGF in the vascular endothelial cell of infarction surrounding zone significantly increases (as shown in Figure 4 C); Although also can be observed the expression of VEGF in astrocyte and neuron, the impact (data do not show) that not processed by tPA.
Because the above results ADAMTS13 that shows to recombinate suppresses the cerebral hemorrhage of tPA induction, this experiment has also detected the rising whether ADAMTS13 that recombinates can reduce the vegf expression of tPA induction, the result of Western Blot experiment shows, restructuring ADAMTS13 significantly suppresses the increase (as shown in Fig. 4 A and B) of the vegf expression that tPA causes, consistent with the above results, in the vascular endothelial cell of infarction surrounding zone, the immunocompetence of the VEGF of tPA induction also significantly reduces (as shown in Figure 4 C).
Existing bibliographical information angiopoietin (Ang)-1 and Ang-2 regulate the permeability 35 of Neurons Against Cerebral Ischemia blood vessel, therefore this measuring the expression of Ang-1 and Ang-2 in cerebral tissue, result shows, the expression of Ang-1 and Ang-2 is basically identical between reason group throughout, shows that Ang-1 and Ang-2 do not participate in regulating the cerebral hemorrhage of tPA induction.
(5) vegf expression of restructuring ADAMTS13 inhibition tPA induction is relevant with Akt path with RhoA
VEGF is regulated by RhoA and Akt path
36,37, and RhoA and Akt participate in regulating the function of blood brain barrier after cerebral ischemia
13,38; For detecting the above-mentioned factor, whether to the VEGF of tPA induction, raise influential, the present embodiment is divided into cerebral ischemia group and tPA processed group by mice, the inhibitor fasudil of intravenous injection RhoA and the inhibitor wortmannin of Akt, result demonstration, fasudil and wortmannin all can suppress the rise of the vegf expression of tPA induction;
Whether the present embodiment has also been measured restructuring ADAMTS13 can suppress the RhoA activation of tPA induction and the phosphorylation of Akt, the result of Western Blot experiment shows, cerebral ischemia obviously increases the activity (as shown in Fig. 5 E and F) of RhoA, and induces the phosphorylation (as shown in Fig. 5 G and H) of Akt; After giving tPA, above-mentioned effect is further expanded, and on the contrary, restructuring ADAMTS13 significantly suppresses the RhoA activation (as shown in Fig. 5 E and F) of tPA induction and the phosphorylation (shown in Fig. 5 G and H) of Akt; The expression of total Akt does not detect difference between each group;
The above results shows, restructuring ADAMTS13 plays by suppressing the activation of RhoA and the phosphorylation of Akt the effect of disturbing tPA-VEGF path.
Claims (5)
1. restructuring ADAMTS13 treats the purposes in cerebral hemorrhage medicine in preparation.
2. restructuring ADAMTS13 is preparing the purposes reducing in the cerebral hemorrhage medicine that after cerebral ischemia, tPA thromboembolism treatment causes.
3. by the purposes described in claim 1 or 2, it is characterized in that, described restructuring ADAMTS13 realizes the inhibition to the vegf expression of tPA induction by RhoA and Akt path.
4. by the purposes described in claim 1 or 2, it is characterized in that, described restructuring ADAMTS13 significantly reduces the activation of RhoA and the phosphorylation of Akt.
5. by the purposes described in claim 1 or 2, it is characterized in that, described restructuring ADAMTS13 increases by suppressing the cerebrovascular permeability of RhoA and Akt mediation, thereby alleviate tPA, the infringement of blood brain barrier is reduced to the cerebral hemorrhage that it causes.
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| WO2011024107A1 (en) * | 2009-08-29 | 2011-03-03 | Semmelweis University | Method for prognosing heart failure |
| JP2012103211A (en) * | 2010-11-12 | 2012-05-31 | Mitsubishi Chemical Medience Corp | Method of detecting the occurrence of liver cancer or predicting risk thereof |
| CN102573792A (en) * | 2009-09-21 | 2012-07-11 | 巴克斯特国际公司 | Stabilized liquid and lyophilized ADAMTS13 formulations |
-
2012
- 2012-07-21 CN CN201210256097.3A patent/CN103566362B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011024107A1 (en) * | 2009-08-29 | 2011-03-03 | Semmelweis University | Method for prognosing heart failure |
| CN102573792A (en) * | 2009-09-21 | 2012-07-11 | 巴克斯特国际公司 | Stabilized liquid and lyophilized ADAMTS13 formulations |
| JP2012103211A (en) * | 2010-11-12 | 2012-05-31 | Mitsubishi Chemical Medience Corp | Method of detecting the occurrence of liver cancer or predicting risk thereof |
Non-Patent Citations (1)
| Title |
|---|
| BING-QIAOZHAO: "von Willebrand factor–cleaving protease ADAMTS13 reduces ischemic brain injury in experimental stroke", 《BLOOD》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016191565A1 (en) * | 2015-05-26 | 2016-12-01 | Baxalta Incorporated | Compositions comprising adamts13 for use in methods for the recanalization of occluded blood vessels in an infarction |
| CN108619496A (en) * | 2017-03-19 | 2018-10-09 | 复旦大学 | ADAMTS13 is preparing the purposes in promoting post-stroke vascular remodeling drug |
| CN110639009A (en) * | 2019-10-24 | 2020-01-03 | 上海市第六人民医院 | Application of recombinant ADAMTS13 in preparation of medicine for treating autoimmune encephalomyelitis |
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| CN103566362B (en) | 2015-07-29 |
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