CN103566362B - Restructuring ADAMTS13 is preparing the purposes in cerebral hemorrhage medicine - Google Patents

Abstract

The invention belongs to the field of biopharmaceuticals, and relates to the use of recombinant ADAMTS13 in the preparation of cerebral hemorrhage drugs, especially the use of recombinant ADAMTS13 in the preparation of drugs for reducing cerebral hemorrhage caused by tPA thrombolysis after cerebral ischemia. The experimental results of the present invention show that the VEGF induced by tPA can be blocked by specific inhibitors of RhoA or Akt under ischemic conditions, while the recombinant ADAMTS13 significantly reduces the activation of RhoA and the phosphorylation of Akt, indicating that the recombinant ADAMTS13 can pass through RhoA and Akt pathway to achieve the inhibition of tPA-induced VEGF expression. The recombinant ADAMTS13 of the present invention reduces the damage of tPA to the blood-brain barrier by inhibiting the increase of cerebrovascular permeability mediated by RhoA and Akt, thereby reducing the cerebral hemorrhage caused by it. Therefore, the combined use of recombinant ADAMTS13 and tPA is to increase thrombolytic A new countermeasure and means of security.

Description

Application of recombinant ADAMTS13 in preparation of cerebral hemorrhage medicine

technical field

The invention belongs to the field of biopharmaceuticals, and relates to the use of recombinant ADAMTS13 in the preparation of cerebral hemorrhage drugs, especially the use of recombinant ADAMTS13 in the preparation of drugs for reducing cerebral hemorrhage caused by tPA thrombolysis after cerebral ischemia.

Background technique

Studies have shown that stroke is one of the main causes of human disability and death; tissue plasminogen activator tPA (Tissue Plasminogen Activator) has become the most effective drug for the treatment of stroke because of its powerful thrombolytic effect; However, the use of tPA for thrombolysis increases the risk of cerebral hemorrhage. Therefore, how to overcome the cerebral hemorrhage caused by tPA is of great significance to reduce the disability and fatality rate of stroke.

The tPA is a serine protease, which plays an important role in maintaining blood coagulation and fibrinolysis; tPA not only regulates the normal central nervous system physiology and neuronal plasticity, but also regulates the central nervous system pathology. plays a key role. The tPA promotes neuron death caused by excitotoxins and increases ischemic brain injury, and knocking out tPA or inhibiting tPA with drugs can significantly protect the ischemic brain. In addition, tPA has been shown to exacerbate ischemic endothelial damage and blood-brain barrier damage by promoting the activation of matrix metalloproteinase-9 (MMP-9) and transcription factor NF-κB, as well as promoting Akt phosphorylation and degradation of extracellular matrix. rupture. A large number of experimental and clinical evidences have shown that the rupture of the blood-brain barrier caused by tPA is one of the most direct causes of cerebral hemorrhage; cerebral hemorrhage.

Studies have shown that von Willebrand factor (VWF) is a complex glycoprotein produced by endothelial cells and megakaryocytes; in addition, ADAMTS13 (a disintegrin-like and metalloprotease withthrombospondin type I repeats-13) is newly discovered in recent years A metalloprotease present in blood plasma, the most important function of the ADAMTS13 is to cleave the newly secreted super large VWF into smaller complex receptor forms with low adhesion, thereby reducing the formation of thrombus; the VWF is ADAMTS13 The only cleavage substrate known so far, in the central nervous system, VWF is abundantly expressed in endothelial cells, however, little is known about the role of VWF in regulating the permeability of the blood-brain barrier. Studies have shown that in ischemic stroke, cerebral hemorrhage and subarachnoid hemorrhage, which are related to the breakdown of the blood-brain barrier, the expression of VWF in plasma is significantly increased; Afterwards, the VWF in the plasma also increased significantly. Recent studies have found that VWF gene deletion has a protective effect on mice with cerebral ischemia. On the contrary, ADAMTS13 gene knockout leads to a significant increase in cerebral ischemic lesions, while recombinant ADAMTS13 can significantly reduce acute cerebral ischemic injury.

So far, there is no report about the use of recombinant ADAMTS13 to reduce cerebral hemorrhage caused by tPA thrombolysis after cerebral ischemia.

References relevant to the present invention are:

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Contents of the invention

The purpose of the present invention is to provide the use of recombinant ADAMTS13 in the preparation of cerebral hemorrhage drugs, especially the use of recombinant ADAMTS13 in the preparation of drugs for reducing cerebral hemorrhage caused by tPA thrombolysis after cerebral ischemia.

The present invention has been proved by experiments that intracerebroventricular injection of VWF causes a rapid increase in the permeability of the blood-brain barrier in non-ischemic mice, and it is also confirmed that VWF lyase ADAMTS13 can block the destructive effect of tPA on cerebral blood vessels after cerebral ischemia, thereby reducing tPA Thrombolysis-induced intracerebral hemorrhage.

Specifically, the present invention has carried out the experiment comprising the following steps:

(1) Intraventricular injection and cerebral vascular permeability measurement;

(2) Animal models of cerebral ischemia;

(3) Behavior measurement;

(4) Measurement of cerebral hemorrhage;

(5) Western blot experiment;

(6) Immunohistochemistry;

(7) Data analysis.

The results show that,

(1) Recombinant ADAMTS13 blocked the increase of blood-brain barrier permeability caused by tPA injection in the non-ischemic brain

Firstly, it was confirmed that injection of tPA into the lateral ventricle of normal mice could increase the permeability of the blood-brain barrier; then non-ischemic mice were injected with VWF through the ventricle, and then injected with Evans blue intravenously, and the brain was perfused 1 hour later to quantitatively detect the blood-brain barrier. Exudation of Evans blue, the results showed that, similar to the injection of tPA, VWF significantly increased the permeability of the blood-brain barrier; however, the combined injection of tPA and VWF caused no significant increase in the permeability of the blood-brain barrier compared with the two alone The differences indicate that tPA and VWF affect the permeability of the blood-brain barrier through the same pathway; further, the results show that administration of 50ng or 100ng recombinant ADAMTS13 in the lateral ventricle can significantly reduce the amount of Evans blue exudation in the brain of mice injected with tPA, However, single injection of recombinant ADAMTS13 had no effect on the permeability of the blood-brain barrier;

(2) Recombinant ADAMTS13 inhibits the damage effect of tPA on the blood-brain barrier after cerebral ischemia

The expression changes of ZO-1 (a major tight junction protein) and collagen IV (a major vascular basement membrane protein) were detected using a 45-minute regional cerebral ischemia-reperfusion model in mice. The results showed that cerebral ischemia Significantly reduced the expression of ZO-1 and moderately reduced the expression of collagen IV; compared with the PBS-treated group, intravenous injection of tPA two hours after cerebral ischemia resulted in a significant reduction of ZO-1 and collagen IV; however, 3 hours after cerebral ischemia The administration of recombinant ADAMTS13 into the lateral ventricle significantly alleviated the degradation of ZO-1 and collagen IV caused by tPA; further, compared with the control group, tPA significantly increased the exudation of Evans blue in the ischemic cerebral hemisphere; the results showed that the injection of recombinant ADAMTS13 completely reversed the degradation caused by tPA. Changes in the permeability of the blood-brain barrier in mice, single use of recombinant ADAMTS13 has no effect on the permeability of the blood-brain barrier after cerebral ischemia;

(3) Effects of recombinant ADAMTS13 on cerebral hemorrhage and neurological function

Quantitative measurement by spectrophotometer showed that injection of tPA 2 hours after cerebral ischemia led to a significant increase in cerebral hemorrhage 24 hours later; intraventricular administration of recombinant ADAMTS13 3 hours after cerebral ischemia inhibited tPA-induced cerebral hemorrhage; administration of recombinant ADAMTS13 alone ADAMTS13 has no significant effect on cerebral hemorrhage;

In addition, the neurological function scores showed that compared with the mice in the PBS-treated group, the mice in the tPA-treated group showed severe neurological deficits and decreased motor functions; while the neurological function scores and motor functions of mice in the recombinant ADAMTS13+tPA group Significantly better than mice given tPA alone;

(4) Recombinant ADAMTS13 reduces the expression of VEGF in vascular endothelial cells induced by tPA

Using Western blot experiments, the results showed that cerebral ischemia significantly increased the expression ^of VEGF compared with the sham group (consistent with previous reports34); compared with cerebral ischemic mice given PBS, administration of tPA caused VEGF expression further increase;

The results of immunohistochemical staining showed that the administration of tPA caused a significant increase in the expression of VEGF in the vascular endothelial cells in the peri-infarct area; although the expression of VEGF could also be observed in astrocytes and neurons, it was not affected by tPA treatment. Influence;

The results of Western blot experiments showed that recombinant ADAMTS13 significantly inhibited the increase of VEGF expression induced by tPA, consistent with this result, the immune activity of VEGF induced by tPA in the vascular endothelial cells in the peri-infarct area was also significantly reduced;

The expression levels of Ang-1 and Ang-2 in the brain tissue were measured, and the results showed that the expressions of Ang-1 and Ang-2 were basically consistent among the treatment groups, indicating that Ang-1 and Ang-2 were not involved in the regulation of tPA-induced cerebral hemorrhage;

(5) The inhibition of tPA-induced VEGF expression by recombinant ADAMTS13 is related to the RhoA and Akt pathways

The mice were divided into the cerebral ischemia group and the tPA treatment group, and the RhoA inhibitor fasudil and the Akt inhibitor wortmannin were injected intravenously. The results showed that both fasudil and wortmannin could inhibit tPA-induced VEGF upregulation of expression;

The results of Western blot experiments showed that cerebral ischemia significantly increased the activity of RhoA and induced the phosphorylation of Akt; when tPA was administered, the above effects were further amplified; on the contrary, recombinant ADAMTS13 significantly inhibited the activation of RhoA and the phosphorylation of Akt induced by tPA; The expression of total Akt was not detected to be different among the groups;

The above results indicated that recombinant ADAMTS13 interfered with the tPA-VEGF pathway by inhibiting the activation of RhoA and the phosphorylation of Akt.

The experimental results of the present invention show that under ischemic conditions, the VEGF induced by tPA can be blocked by specific inhibitors of RhoA or Akt, while recombinant ADAMTS13 significantly reduces the activation of RhoA and the phosphorylation of Akt, indicating that recombinant ADAMTS13 has a strong effect on tPA Inhibition of induced VEGF expression is achieved through the RhoA and Akt pathways.

The recombinant ADAMTS13 of the present invention reduces the damage of tPA to the blood-brain barrier by inhibiting the increase of cerebrovascular permeability mediated by RhoA and Akt, thereby reducing the cerebral hemorrhage caused by it. Therefore, the combined use of recombinant ADAMTS13 and tPA is to increase thrombolytic A new countermeasure and means of security.

Description of drawings

Figure 1 shows that under non-ischemic conditions, recombinant ADAMTS13 inhibits the increase in the permeability of the blood-brain barrier caused by tPA, wherein,

A: After intracerebroventricular injection of PBS, tPA, VWF or tPA plus VWF, the measured value of Evans blue exudation in the brain 1 hour after intravenous injection of Evans blue;

B: After intravenous injection of PBS, tPA, combined injection of tPA and 50 or 100ng recombinant ADAMTS13, single injection of recombinant ADAMTS13, the measured value of Evans blue exudation in the brain after intravenous injection of Evans blue for 1 hour;

Values are mean + standard deviation, n=7-8 for each group, *P<0.05.

Figure 2 shows that recombinant ADAMTS13 inhibits tPA-induced blood-brain barrier breakdown after cerebral ischemia, wherein,

A, C: 24 hours of cerebral ischemia, sham operation group and control group, tPA administration group, tPA+recombinant ADAMTS13 administration group, representative western blot photos of zo-1 and collagen IV;

B, D: Quantitative determination of ZO-1 and collagen IV in each treatment group; the value adopts mean + standard deviation, n=4 in the sham operation group, n=6 in other groups, *P<0.05;

E: Representative graphs of Evans blue exudation in the control group, tPA administration group, tPA+recombinant ADAMTS13 administration group and recombinant ADAMTS13 administration group alone at 24 hours of cerebral ischemia;

F: Quantitative analysis of Evans blue exudation in each treatment group; the value adopts mean + standard deviation, n=7 for each group, *P<0.05.

Figure 3 shows the effect of recombinant ADAMTS13 on neurological function after cerebral hemorrhage and cerebral ischemia, wherein,

A: Representative dorsal and coronal sections of the brain showing intracerebral hemorrhage at 24 hours of cerebral ischemia. The pictures are from mice in the control group, tPA administration group, tPA+recombinant ADAMTS13 administration group and recombinant ADAMTS13 administration group ; What the arrow points to is the massive hemorrhage in the cerebral ischemic area of the mice given tPA;

B: Quantitative detection results of cerebral hemorrhage with a spectrophotometer; the value is the mean + standard deviation, n=8 for each group, *P<0.05. (C) Neurological function score and (D) Motor behavior of mice in the control group, tPA administration group, tPA+recombinant ADAMTS13 administration group and recombinant ADAMTS13 administration group alone at 24 hours of cerebral ischemia; the values are mean + standard deviation , each group n=8, *P<0.05.

Figure 4 shows that recombinant ADAMTS13 reduces tPA-induced VEGF expression in vascular endothelial cells, wherein,

A: 24 hours of cerebral ischemia, representative western blot pictures of VEGF expression in mice in sham operation group, control group, tPA administration group, tPA+recombinant ADAMTS13 administration group and recombinant ADAMTS13 administration group alone;

B: Quantitative measurement results of VEGF expression in each treatment group; the value adopts mean + standard deviation, n=4 in the sham operation group, n=6 in other groups, *P<0.05;

C: Immunohistochemical confocal images of VEGF (dark) and endothelial cells (CD31, bright) in the peri-infarct area of mice given tPA group or tPA+recombinant ADAMTS13 group after 24 hours of cerebral ischemia; asterisks indicate vascular lumen; scale bar: 15 μm ;

D, E: Representative western blot pictures of Ang-1 and Ang-2 expression in sham operation group mice, control group, tPA administration group, tPA+recombinant ADAMTS13 administration group after 24 hours of cerebral ischemia;

F, G: Quantitative measurement results of Ang-1 and Ang-2 expression in each group; the values are mean + standard deviation, n=6 in each group, *P<0.05.

Figure 5 shows that the inhibition of tPA-induced VEGF expression by recombinant ADAMTS13 is related to the RhoA and Akt pathways, wherein,

A, C: Representative western blot pictures of VEGF expression in the ischemic brain of mice given tPA group, tPA+wortmannin or fasudil group after 24 hours of cerebral ischemia;

B, D: Quantitative measurement results of VEGF expression in each group; the value is the mean + standard deviation, each group n=5, *P<0.05;

E, F: Representative western blot pictures of phosphorylated Akt, total Akt and RhoA in mice in sham operation group, control group, tPA administration group, tPA+recombinant ADAMTS13 administration group after 24 hours of cerebral ischemia;

G, H: Quantitative determination results of phosphorylated Akt and RhoA in each group; the values are mean + standard deviation, n=4 in the sham operation group, n=6 in other groups, *P<0.05.

specific embodiment

Example 1

1. Materials and methods

(1) Reagents and antibodies

Evans blue, Drabkin, paraformaldehyde, D-glucose, formamide, wortmannin (purchased from Sigma-Aldrich) (St Louis, MO, USA), human recombinant tPA (Aitongli) was purchased from BoehringerIngelheim (Mannheim, Germany ), human VWF protein was purchased from Haematologic Tecnologies (EssexJunction, VT, USA), human recombinant ADAMTS13 was purchased from R&D systems (neapolis, MN, USA), and fasudil was purchased from Calbiochem (San Diego, CA, USA). Primary antibodies include rabbit anti-zonulaoccludens (ZO-1) (Invitrogen, Camarillo, CA, USA), rabbit anti-collagen IV (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-VEGF (vascular endothelial growth factor) (Abcam, MA, USA), goat anti-Angiopoietin (Ang)-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-Ang-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti -phospho-Akt (serine 473) (Cell Signaling Tecnology, Beverly, MA, USA), rabbit anti-Akt (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) ) rat anti-CD31 (PECAM-1, BD Pargen, San Diego, CA, USA), rabbit anti-β-actin (Cell Signaling technology, Beverly, MA, USA). Secondary antibodies include: Alexa Fluor 594donkey anti-rabbit immunoglobulin G (IgG), Alexa Fluor 488 donkey anti-rat IgG (Invitrogen, Camarillo, CA, USA).

(2) Intraventricular injection and measurement of cerebral vascular permeability

All experiments were approved by the Animal Care and Use Committee of Shanghai Medical College, Fudan University. The present invention uses male adult C57 mice (provided by Shanghai Slack Experimental Animal Co., Ltd., Shanghai, China).

The mice were placed in a stereotaxic instrument (Narisige Scientific Instrument Laboratory, Tokyo, Japan), anesthetized continuously with a gas mixture containing 1-1.5% isoflurane and 30% oxygen, at the following coordinates: 0.2 mm behind the anterior bregma, side opening 1.0mm, ventral depth 3mm, intracranial injection of 3μl PBS, tPA (585ng), VWF (540ng), tPA and VWF, tPA and recombinant ADAMTS13 (50 or 100ng) or recombinant ADAMTS13 (100ng), the injection time is 15 minutes Then, intravenous injection of Evans blue dye (4%, dissolved in PBS, 4ml/kg), 60 minutes later, the mice were perfused with PBS through the heart, and the brain tissue was soaked in formamide solution for 72 hours; using a spectrophotometer (ThermoBioMate 3S , Thermo Scientific, USA) at 620nm wavelength to measure the extravasation of Evans blue in brain tissue.

(3) Animal cerebral ischemia model

The cerebral ischemia model (MCAO) was made according to the method of the prior ^art25,27 , that is, after the male mice were anesthetized with a mixed gas containing 1-1.5% isoflurane, 30% oxygen, and 70% nitrogen, they were injected through the right side of the neck. The middle cerebral artery was blocked by inserting a 7.0 nylon tether with a silicone-coated end into the artery; after 45 minutes of ischemia, the tether was withdrawn for reperfusion; a heating plate (WorldPrecision Instruments, Sarasota, FL, USA) was used to maintain the body temperature of the mouse at 37±0.5 ℃; the embolism and reperfusion of the middle cerebral artery were confirmed by laser Doppler flowmeter (Perimed, Stockholm, Sweden); in order to increase the amount of cerebral hemorrhage, mice were given intraperitoneal injection of 50% D-glucose after 15 minutes of cerebral ischemia (6ml/kg) ^28,29 ; 2 hours after cerebral ischemia, inject 1mg/kg (bolus) tPA (10mg/kg) or PBS intravenously with a micro-injection pump (World Precision Instruments, Sarasota, FL, USA), and the remaining 9 mg/kg was slowly infused over 30 minutes; recombinant ADAMTS13 (100 ng dissolved in 3 μl PBS) or PBS was administered through the right lateral ventricle 3 hours after cerebral ischemia; wortmannin (15 μg/kg dissolved in 100 μl containing 1% dimethyl DMSO) or 1% dimethyl sulfoxide in normal saline before cerebral ischemia; fasudil (10 mg/kg dissolved in 100 μl normal saline) or normal saline in Intravenous injection 30 minutes after cerebral ischemia; 23 hours after cerebral ischemia in mice, intravenous injection of 2% Evans blue (2ml/kg); the amount of Evans blue extravasation in the cerebral hemisphere of the ischemic side was measured by the method described above .

(4) Behavior measurement

24 hours after cerebral ischemia, the neurological damage of mice was scored by double-blind method:

0 points, no neurological impairment;

1 point, inability to fully extend the left front paw;

2 points, the resistance to thrust is weakened;

3 points, turn left spontaneously;

4 points, lack of voluntary activity or unconsciousness.

In the motor behavior test, the mice were put into a test box of 27.3×27.3×40cm (Med Associates, USA), and the distance traveled by the mice within 30 minutes was automatically recorded by an infrared camera, and the SOD-811 analysis software (Med Associates, USA) was used to analyze the distance. Associates Inc., St Alban's, VT, USA) analyzed the data.

(5) Measurement of cerebral hemorrhage

24 hours after ischemia, inject excess chloral hydrate into the mice, and perfuse the heart with PBS; collect the brain tissue on the ischemic side, add 500 μl Drabkin solution, homogenate, and centrifuge at 13,000 rpm at 4°C for 30 minutes; collect the supernatant and use spectrophotometer A photometer (Thermo BioMate 3S, Thermo Scientific, USA) measured the optical density at a wavelength of 540nm; a standard curve was prepared using homologous mouse blood, and the amount of bleeding corresponding to the optical density value was calculated according to the standard curve25,30.

(6) Western blot experiment

The brain tissue of the ischemic side and the corresponding brain tissue of the sham operation group were lysed with lysate; the brain tissue was homogenized and centrifuged in RIPA lysate (Millipore, Billerica, MA, USA) containing protease inhibitor cocktails, and then washed with BCA protein assay (Thermo BioMate 3S, Thermo Scientific, USA) was used to detect the protein concentration; equal amounts of protein samples were subjected to 8%, 10% or 12% Tris-glycine gel electrophoresis, respectively, and then transferred to PVDF membrane; using 1XTBST 5% skimmed milk was used to block at room temperature for 1 hour, and the primary antibody was incubated overnight at 4°C. After rinsing, the secondary antibody was added and incubated at room temperature for 1 hour; Bio-Rad, USA) for scanning and quantitative analysis; β-actin was used as an internal reference.

(7) Immunohistochemistry

24 hours after cerebral ischemia, the mice were deeply anesthetized with an excessive amount of chloral hydrate, then perfused with PBS and 4% paraformaldehyde solution in turn, and the brain was taken; the brain tissue was dehydrated with 30% sucrose; the brain was sliced coronally (20 μm) Block with PBS blocking solution containing 1% bovine serum albumin and 5% normal donkey serum, and incubate overnight at 4°C with rabbit anti-VEGF and rat anti-CD31 antibody mixture; after rinsing the sections, use Alexa Fluor 594 donkey anti-rabbit The mixture of IgG and Alexa Fluor 488 donkey anti-rat Ig was incubated; the images were obtained using Olympus FV1000 confocal microscope, and the FV10-ASW software was used for image processing.

(8) Data analysis

The data are expressed as mean ± SD, and statistical analysis is performed by Boneferroni multi-group comparison after one-way analysis of variance; statistical analysis of behavioral data is performed by Dunn multi-group comparison after Kruskal–Wallis test analysis; Paired two-tailed Student's t test. P<0.05 was considered to be statistically different.

2. Results

(1) Recombinant ADAMTS13 blocked the increase of blood-brain barrier permeability caused by tPA injection in the non-ischemic brain

Firstly, it was confirmed that the intracerebroventricular injection of tPA in normal mice could increase the permeability of the blood-brain barrier (as shown in Figure 1A) ^28,31 ; in order to test whether VWF could also induce the increase of the permeability of the blood-brain In mice, VWF was injected into the ventricle, and then Evans blue was injected intravenously. After 1 hour, the brain was perfused, and the amount of Evans blue exudation in the brain was quantitatively detected; the results showed that, similar to the injection of tPA, VWF significantly increased the permeability of the blood-brain barrier (as shown in Figure 1A); however, the combined injection of tPA and VWF caused no significant difference in the degree of increased BBB permeability compared with the two alone, suggesting that tPA and VWF affect BBB permeability through the same pathway ; Further research on whether the VWF lyase ADAMTS13 can inhibit the damage of the blood-brain barrier caused by tPA, the results showed that administration of 50ng or 100ng recombinant ADAMTS13 in the lateral ventricle can significantly reduce the amount of Evans blue exudation in the brain of mice injected with tPA (as shown in the figure 1B); since 100 ng of recombinant ADAMTS13 was more effective, 100 ng of recombinant ADAMTS13 was used in subsequent experiments; injection of recombinant ADAMTS13 alone had no effect on the permeability of the blood-brain barrier.

(2) Recombinant ADAMTS13 inhibits the damage effect of tPA on the blood-brain barrier after cerebral ischemia

The expression changes of ZO-1 (a major tight junction protein) and collagen IV (a major vascular basement membrane protein) were detected using a 45-minute regional cerebral ischemia-reperfusion model in mice. The results showed that cerebral ischemia The expression of ZO-1 was significantly reduced (as shown in Figure 2A and B), and the expression of collagen IV was moderately reduced (as shown in Figure 2C and D); compared with the PBS-treated group, intravenous injection of tPA two hours after cerebral ischemia resulted in Significant reductions in ZO-1 (shown in Figure 2A and B) and collagen IV (shown in Figure 2C and D); however, intracerebroventricular administration of recombinant ADAMTS13 3 hours after cerebral ischemia significantly attenuated tPA-induced ZO-1 and Degradation of collagen IV; the results also showed that compared with the control group, tPA significantly increased the exudation of Evans blue in the ischemic brain hemisphere (as shown in Figure 2E and F), and injection of recombinant ADAMTS13 completely reversed the blood-brain barrier of mice caused by tPA Permeability changes (as shown in Figure 2E and F); while recombinant ADAMTS13 alone had no effect on the permeability of the blood-brain barrier after cerebral ischemia.

(3) Effects of recombinant ADAMTS13 on cerebral hemorrhage and neurological function

Quantitative measurement by spectrophotometer showed that infusion of tPA 2 hours after cerebral ischemia resulted in a significant increase in cerebral hemorrhage after 24 hours (as shown in Figure 3A and B), and intraventricular administration of recombinant ADAMTS13 inhibited 3 hours after cerebral ischemia. tPA-induced cerebral hemorrhage (as shown in Figure 3A and B); administration of recombinant ADAMTS13 alone had no significant effect on cerebral hemorrhage.

In addition, the neurological function scores showed that compared with the mice in the PBS-treated group, the mice in the tPA-treated group showed severe neurological function loss and decreased motor function; the neurological function scores and motor function of mice in the recombinant ADAMTS13+tPA group were significantly Better than mice given tPA alone.

(4) Recombinant ADAMTS13 reduces the expression of VEGF in vascular endothelial cells induced by tPA

The results of Western blot experiments showed that compared with the sham group, cerebral ischemia significantly increased ^the expression of VEGF, which was consistent with previous reports34; compared with the cerebral ischemic mice given PBS, administration of tPA caused the expression of VEGF further increase (as shown in Figure 4A and B);

Immunohistochemical staining results showed that administration of tPA caused a significant increase in the expression of VEGF in vascular endothelial cells in the peri-infarct zone (as shown in Figure 4C); although VEGF expression was also observed in astrocytes and neurons , but was not affected by tPA treatment (data not shown).

Since the above results show that recombinant ADAMTS13 inhibits tPA-induced cerebral hemorrhage, this experiment also tested whether recombinant ADAMTS13 can reduce the increase in VEGF expression induced by tPA. The results of Western blot experiments showed that recombinant ADAMTS13 significantly inhibited the increase in VEGF expression induced by tPA (As shown in Figure 4A and B), consistent with the above results, the immune activity of tPA-induced VEGF in vascular endothelial cells in the peri-infarct area was also significantly reduced (as shown in Figure 4C).

Existing literature reports that angiopoietin (Ang)-1 and Ang-2 regulate the permeability of cerebral vessels after ischemia35, so this experiment measured the expression levels of Ang-1 and Ang-2 in brain tissue, and the results showed that Ang-1 The expressions of Ang-1 and Ang-2 were basically consistent among the treatment groups, indicating that Ang-1 and Ang-2 were not involved in the regulation of tPA-induced intracerebral hemorrhage.

(5) The inhibition of tPA-induced VEGF expression by recombinant ADAMTS13 is related to the RhoA and Akt pathways

VEGF is regulated by the RhoA and Akt pathways ^36,37 , and both RhoA and Akt are involved in regulating ^the function of the blood-brain barrier after cerebral ischemia; Rats were divided into cerebral ischemia group and tPA treatment group, and RhoA inhibitor fasudil and Akt inhibitor wortmannin were injected intravenously. The results showed that both fasudil and wortmannin could inhibit the expression of VEGF induced by tPA. raised;

This embodiment also determined whether recombinant ADAMTS13 could inhibit tPA-induced activation of RhoA and phosphorylation of Akt. The results of Western blot experiments showed that cerebral ischemia significantly increased the activity of RhoA (as shown in Figure 5E and F), and induced Akt (as shown in Figure 5G and H); when tPA was administered, the above effects were further amplified, on the contrary, recombinant ADAMTS13 significantly inhibited tPA-induced activation of RhoA (as shown in Figure 5E and F) and Akt phosphorylation ( Shown in Figure 5G and H); the expression of total Akt was not detected to be different among the groups;

The above results indicated that recombinant ADAMTS13 interfered with the tPA-VEGF pathway by inhibiting the activation of RhoA and the phosphorylation of Akt.

Claims (5)

1. The use of recombinant ADAMTS13 in the preparation of drugs for the treatment of cerebral hemorrhage. 2. The use of recombinant ADAMTS13 in the preparation of a drug for reducing cerebral hemorrhage caused by tPA thrombolytic therapy after cerebral ischemia. 3. The use according to claim 1 or 2, characterized in that the recombinant ADAMTS13 suppresses tPA-induced VEGF expression through RhoA and Akt pathways. 4. The use according to claim 1 or 2, characterized in that said recombinant ADAMTS13 significantly reduces the activation of RhoA and the phosphorylation of Akt. 5. The use according to claim 1 or 2, characterized in that said recombinant ADAMTS13 reduces the damage of tPA to the blood-brain barrier by inhibiting the increase of cerebrovascular permeability mediated by RhoA and Akt, thereby reducing the brain damage caused by it. bleeding.