CN111481668A - Application of macrophage migration inhibitory factor as drug target in treating perioperative stroke - Google Patents

Abstract

The invention discloses application of macrophage migration inhibitory factor as a drug target in treating perioperative stroke. The invention discovers that MIF-mediated endothelial cell apoptosis is a main mechanism for destroying the integrity of a blood brain barrier and the neurological function defect after stroke after PIS for the first time, and the MIF-mediated endothelial cell apoptosis is used as a drug target for treating the perioperative stroke. The invention discloses a mechanism of BBB damage after perioperative stroke and a relation between the mechanism and peripheral immune response, finds an innovative mechanism for the nerve function damage of perioperative ischemic stroke, and provides an innovative treatment idea for improving the neurobehavioral function and cognitive function of perioperative stroke.

Description

Application of macrophage migration inhibitory factor as drug target in treating perioperative stroke

Technical Field

The invention belongs to the field of medicines, and relates to application of a macrophage migration inhibitory factor as a medicine target in treating perioperative stroke.

Background

Perioperative ischemic stroke is a great clinical problem for clinical anesthetists, has the characteristics of high incidence, great harm, troublesome treatment and the like, and is more and more concerned due to the high incidence and destructive consequences of the perioperative ischemic stroke in surgical patients. The incidence of perioperative stroke (PIS) is closely related to the type and complexity of the surgical procedure, with a reported incidence of PIS in postoperative patients as high as 2.9% -4%. Besides the heart operation, the perioperative stroke incidence rate is higher in the general surgical operation with extremely high operation amount, and the cognitive function decline risk is obviously increased after 1 year.

The Blood Brain Barrier (BBB), which is composed of endothelial cells, astrocytes and pericytes, plays an important role in maintaining normal neural function, and dysfunction thereof has been demonstrated to be a core mechanism of nervous system deterioration after stroke. Degeneration or apoptosis of endothelial cells plays a key role in BBB injury, and is one of the causes of poor prognosis of stroke. Surgical trauma can induce rapid activation of immune cells. Among them, mononuclear macrophages are an important cell type in the innate immune system that recognizes tissue damage. Surgery has been reported to cause peripheral macrophages to secrete macrophage Migration Inhibitory Factor (MIF). MIF is a multifunctional protein with a broad immunomodulatory function, which is produced and stored in cytoplasmic capsules and can be rapidly released in response to a series of stimuli, and in recent years, the complex function of MIF has been receiving increasing attention. After the MIF is released, the MIF not only can be used as a cytokine to induce inflammatory reaction, but also has the activities of protease and endonuclease to induce cell DNA damage and apoptosis. However, the role of MIF in BBB damage after PIS has not been reported.

Disclosure of Invention

Epidemiological studies have shown that surgery is associated with an increased risk of stroke. However, the molecular mechanisms of how surgery affects stroke remain unknown. The invention aims to provide a drug target for treating perioperative stroke.

In order to achieve the purpose, the invention provides application of macrophage migration inhibitory factor as a drug target in treating perioperative stroke.

The invention also provides application of the macrophage migration inhibitory factor antagonist in preparing a medicament for treating the stroke in the perioperative period.

Preferably, the macrophage migration inhibitory factor antagonist is ISO-1.

The invention also provides application of the CD74 receptor as a drug target in treating perioperative stroke.

The invention also provides application of the CD74 receptor blocker in preparing a medicament for treating the perioperative stroke.

The invention utilizes the far-end middle cerebral artery blockage combined tibia fracture post-internal fixation animal model to simulate perioperative stroke, and proves that the post-stroke brain injury and blood brain barrier injury of perioperative stroke mice are more serious compared with single stroke. 3 days after stroke, the perioperative stroke mouse has obviously increased cerebral infarction volume and blood brain barrier permeability compared with a single stroke mouse. Through nerve function assessment, the nerve function damage of perioperative stroke mice after operation is found to be more serious. In our study, in the case of ischemic stroke, damage caused by endothelial cells forming the blood brain barrier disrupts the structure and function of the neurovascular unit. Structural deformation of brain microvascular endothelial cells may be the cause of rupture of the BBB in the early stages of ischemic stroke, leading to increased permeability of the BBB.

According to our study, apoptosis of brain endothelial cells was significantly increased in perioperative stroke mice. In order to further explore the apoptosis form of endothelial cells after perioperative stroke, through an immunofluorescence technique, we find that the expression of a marker PARP-1 representing DNA single-strand damage is increased, and the expression of markers gamma H2AX and Puma representing DNA double-strand apoptosis has no obvious change in endothelial cells of perioperative stroke mice and pure stroke mice, which indicates that the brain endothelial cell apoptosis is mainly caused by single-strand DNA damage after perioperative stroke. Our data show that macrophages are widely activated and release large amounts of MIF in peripheral blood one day post-surgery in perioperative patients. In the mouse experiment, peripheral macrophages were activated and induced MIF release in response to a danger signal 3 days after perioperative stroke, consistent with previous studies showing that surgery allows peripheral macrophages to secrete MIF. By flow-based techniques, we have analyzed different factors individually and have found that surgical trauma plays a major role in inducing macrophages to release MIF.

MIF promotes the production of proinflammatory cytokines, including TNF- α, I L-1 β and I L-6, which have been reported to increase BBB permeability, after MIF release, not only can induce an inflammatory response as a cytokine, but also have protease and endonuclease activity, to test the causal relationship between MIF and BBB injury in perioperative stroke, we have found that rMIF treatment can exacerbate post-stroke infarct volume and BBB permeability by intravenous administration of rMIF and its antagonist ISO-1, and that simultaneous administration of ISO-1 can alleviate the above-mentioned phenomena.

To verify whether exogenous or endogenous MIF plays a major role in endothelial apoptosis in PIS mice, we administered both rMIF and ISO-1 in PIS and stroke alone (IS) mice. Exogenous MIF increased MIF expression. Similar to the in vivo experiments, exogenous MIF increased MIF expression in bned 3 cells in vitro after OGD/reoxygenation. Furthermore, mRNA expression of MIF was found to be unchanged in different groups after OGD/reoxygenation using qPCR. Taken together, these results demonstrate that exogenous MIF plays a major role in BBB disruption and endothelial apoptosis in PIS mice.

Endothelial cells can be activated by inflammatory factors secreted by macrophages and promote the entry of inflammatory factors into cells by endocytosis. During endocytosis, the cell membrane is invaginated to form a vesicle, and extracellular molecules are captured in the vesicle and enter the cell. In our study, we found that expression of CD74 was increased in PIS mouse endothelial cells compared to IS mice. Meanwhile, knockout of CD74 in bned 3 cells after OGD prevents MIF endocytosis. This is the first demonstration that peripheral MIF enters endothelial cells by endocytosis, which is mediated by CD74 receptors in endothelial cells after PIS.

The invention has the beneficial effects that:

the invention discovers that the surgical wound activates peripheral macrophages and releases MIF for the first time, and the peripheral MIF enters endothelial cells through endocytosis mediated by CD74 receptors, exerts endonuclease activity thereof and aggravates SSB of endothelial cells of PIS mice. The results further reveal the mechanism of BBB damage after perioperative stroke and the relation between the mechanism and peripheral immune response, find an innovative mechanism for the nerve function damage of perioperative ischemic stroke, and provide an innovative treatment idea for improving the neurobehavioral function and cognitive function of perioperative stroke.

Drawings

FIG. 1 perioperative stroke mice (PIS) are compared to ZO-1 expression levels in stroke alone (IS) mice on a scale of 20 μm.

FIG. 2A.2,3, 5-Triphenyltetrazolium chloride (TTC) stained representative images of brain sections showing infarcted areas (left) of 7 consecutive coronal sections (1 mm apart) on days 1 and 3 after administration of rMIF or ISO-1 in perioperative stroke mice (PIS) and in stroke-only mice (IS); infarct volume was quantified by TTC staining on days 1 and 3 after administration of rMIF or ISO-1 in perioperative stroke mice (PIS) and stroke alone mice (IS) (right, n-4-7);

FIG. 2B shows that in perioperative stroke mice (PIS) and stroke alone mice (IS), rMIF worsens and ISO-1 improves IgG extravasation (left) following dMCAO; immunofluorescent staining of mouse IgG determined the quantification of endogenous IgG leakage MFI ((right, n-6/group);

comparison of the immunofluorescent-stained ZO-1 expression levels in Non-OGD, OGD + MIF + ISO-1(OGD + M + I) groups in ben 3 cells at a scale of 100 μ M;

figure 2d. TUNE L and CD31 double immunostaining in perioperative stroke mice (PIS) and stroke alone mice (IS) brain endothelial cells after rMIF or ISO-1 treatment (left) and TUNE L positive cells along the microvasculature quantified in the brain (right) on a scale of 100 μm.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

The present invention determines sample size by performing power calculations of mean square deviation and standard deviation on the main parameters (power 80%, α ═ 0.05). consecutive data are expressed as mean ± Standard Error (SEM). two sets of data were analyzed using the t-test. three or more sets of data were analyzed using one-way analysis of variance and multiple comparisons were performed using the Bonferroni test.

Firstly, a mouse cerebral ischemia model:

all animal experiments were approved by the institutional animal care and use committee of the care and use facility of laboratory animals, and were performed according to the guidelines for care and use of laboratory animals, male 8 to 10 week old C57/B L6 mice were selected to establish a model of distal stroke (dmco), mice were anesthetized with 2% isoflurane, skin incisions were made in the neck, the left internal carotid artery was exposed and ligated, after the neck incision was closed, another skin incision was made between the left eye and the ear, the temporal muscle was dissected and a bore was opened to expose the distal portion of the middle cerebral artery, then dura was dissected and the distal middle cerebral artery was coagulated by low intensity bipolar coagulation (shanghai picnic co electronic limited) directly outside the spina bifida.

II, a tibia fracture model:

tibia fracture internal fixation was performed 24 hours before the cerebral ischemia model was performed to establish a perioperative stroke model, and the mice were anesthetized with 2% isoflurane. Animals received right hind limb open tibial fractures and underwent intramedullary fixation under sterile surgical conditions 6 rectal temperature was maintained at 37 ± 0.5 ℃ throughout the procedure using a heat blanket. Control mice for fracture received hind limb hair shaving.

Three, Doppler blood flow detection

Mice were induced with a mixture of gases containing 3% isoflurane (N2O: O2 ═ 67%: 30%) until no physical response to tail pressure, and then anesthesia was maintained with 1.5% isoflurane. The black hair at the top of the head was shaved off and leaked out of the scalp before the mice were molded. After iodophor sterilization, a 1.5 cm incision was made to expose the skull. The broken hair on the craniofacial surfaces was removed with a wet cotton swab. The skull is exposed within the monitored area. And setting parameters. Changes in cerebral blood flow over time were recorded and expressed as a percentage of the contralateral MCAO baseline. After monitoring, the scalp was closed. The mouse is subjected to ischemic stroke model construction, and a wire plug is placed at the middle cerebral artery. And recording the cerebral tissue blood perfusion immediately after the model is successfully established. Brain tissue blood flow was recorded 1 hour and 1 day after removal of the wire plug.

Doppler blood flow meter parameters

Tetrakis, 2,3, 5-triphenyltetrazolium chloride staining (TTC staining)

TTC staining was used to determine infarct volume. For TTC staining, coronal sections were prepared 1mm from the forehead and sections were immersed in 1.5% TTC (Sigma-Aldrich) for 20 minutes at 37 ℃, infarct volume was determined by the area without lateral TTC staining. We scanned the stained sections and measured the infarct area using ImageJ. Total infarct volume was obtained by integrating the measured infarct area and the distance between the sections. The reduction of the contralateral hemisphere volume was subtracted ipsilaterally to account for the effects of cerebral edema.

After a perioperative ischemic stroke model is established, local cerebral blood flow (rCBF) is detected before and after dMCAO through laser speckle two-dimensional imaging. Compared with the pure stroke mice, the rCBF of the perioperative stroke mice is obviously reduced by dMCAO 3 days, and the rCBF is not statistically different 1 day after the dMCAO. The TTC staining is used for measuring ischemic cerebral infarction, and the perioperative cerebral apoplexy mouse has obviously increased infarct volume 3 days after dMCAO compared with a single cerebral apoplexy mouse. Consistent with rCBF, the cerebral infarctions of the two groups did not show significant differences 1 day after dMCAO.

Fifth, evaluation of neurological function

We studied the effect of post-dcao surgery on sensorimotor performance using three different behavioral tests, including modified Garica scores, staggered tests and taped tests.

We scored each test from 0 to 3 (max score 15) (a) proprioception, (b) forelimb walking, (c) limb symmetry, (d) lateral rotation, (e) climbing in a staggered experimental test, mice were placed on overhead steel grids, then foot defect errors were recorded during locomotion (when the animal's forelimb was staggered and then dropped through the grid), in a torn tape test, two identical pressure tapes were applied to each paw (0.3 × 0.4cm 2) tape placement sequence (right or left) alternated between each animal and each treatment session.

The results show that:

perioperative stroke mice have proprioception, forelimb walking, symmetric limbs, lateral turning, climbing and total score obviously lower than that of simple stroke mice (P <0.001, P <0.01, P <0.001, P < 0.0001).

In the staggered step experiment, perioperative stroke mice developed more pronged foot on days 3 and 7 after stroke (P <0.05, P <0.01, respectively).

In the tape-stripping experiment, on days 7 and 14 after stroke, perioperative stroke mice spent longer time to remove the sticky paper on their soles (P <0.0001, P <0.05, respectively) compared to stroke-only mice. Taken together, these results demonstrate that perioperative stroke mice exhibit severe neurological dysfunction both early and late in stroke.

Evans Blue (EB) detection

To study BBB destruction after perioperative stroke, we examined the amount of infiltration into brain parenchyma by EB and IgG assays.

3 hours before mice were sacrificed, Evans blue (4% in PBS, 4ml/kg, Sigma-Aldrich) was injected intravenously. N, N-dimethylformamide was added to brain tissue and the tissue was sonicated on ice for 1 minute. Evans blue levels in each hemisphere were determined using the following formula: { A620nm- [ (A500nm + A740nm)/2] }/mg wet weight.

We found that EB and IgG extravasation was significantly increased in perioperative stroke mice 3 days post dMCAO (FIG. 2B), but there was no difference 1 day post dMCAO.

Magnetic resonance imaging technology for cranium and brain of seven, small animals

T2-weighted imaging of all mice was performed using a T2 spin echo sequence (repetition time/echo time 4000/33 ms.) with a T2-weighted imaging field of view of 35 × 35mm, the number of excitations was 2, the matrix was 256 × 256mm twenty-five 1.0mm thick coronal slices were obtained from each animal after 10 pre-contrast images were obtained, a high dose of contrast agent was injected manually through the caudal vein of each mouse at 0.2mmol/kg for 10 seconds, Bei L u pharmacological Co., L td., Beijing, China) 120 dynamic T1 spin echo images were acquired with parameters of repetition time/echo time of 200/10.5ms, field of view, 30x 30 mm, number of excitations, 3, flip angle, 178.5 degrees, slice thickness, 2mm, and areal resolution of 234 μm.

T2 weighted imaging (T2WI) and dynamic contrast enhanced MRI (DCE) are used for detecting infarct volume and BBB permeability of perioperative stroke mice and pure stroke mice, and the results show that the perioperative stroke mice have higher Ktrans maps, RVE and RVP values than the pure stroke mice, and T2WI indicates that the BBB permeability of PIS mice after dMCAO is obviously improved.

Eighth, immunofluorescent staining and quantitation, Western blotting

To assess the integrity of the BBB, we examined the expression of the tight junction protein using immunofluorescence staining and Western blot.

Immunostaining was performed on floating 20 μm sections. Sections were incubated overnight at 4 ℃ in the following antibodies: goat anti-CD 31(R & D), rabbit anti-ZO-1 (Proteintech), rabbit anti-MIF (Abcam), rabbit anti-PARP-1 (Abcam), rabbit anti-H2 AX (Abcam), rabbit anti-Biao horse (Abcam), goat anti-GFAP (Abcam), goat anti-CD 13(R & D). Images were captured using fluorescence microscopy (Olympuc) or confocal microscopy (Olympus Fluoview FV3000, Olympus, pennsylvania central valley). The immune positive cell count is expressed as the average number of cells per millimeter of blood vessel. Three randomly selected microscopic regions of each cerebral cortex in three serial sections of each brain were analyzed by the investigator.

As shown in FIG. 1, 3 days after stroke, perioperative stroke (PIS) mice had significantly lower levels of zonulin ZO-1 than stroke alone (IS), indicating that perioperative stroke blood brain barrier integrity was more severely compromised.

Nine, detection of Single-stranded DNA Damage and TUNE L staining of brain sections

Since apoptosis of cells following ischemic stroke is an important cytopathological basis for ischemic brain injury, we used immunofluorescent staining to detect apoptosis of different cells that make up the vascular unit.

Sections were air dried, fixed with 10% formalin for 10 minutes, and washed three times in PBS. The sections were then fixed in ethanol/acetic acid (2: 1, vol/vol) for 5 min and washed three times in PBS. After sections were permeabilized with 1% Triton X-100 for 20 min and quenched with 2% H2O2 for 15 min, they were washed three times in PBS. Then, the sections were incubated in a wet air chamber at 37 ℃ for 90 minutes at 30. mu.M with a PANT reaction mixture containing 5mM MgCl 2,10mM 2-mercaptoethanol, 20. mu.g/ml bovine serum albumin, dGTP, dCTP and dTTP. Each, 29. mu.M biotinylated dATP, 1. mu.M dATP and 40U/ml E.coli DNA polymerase I (Sigma) in PBS (pH 7.4). The reaction was stopped by two PBS washes. After 5 minutes of washing in PBS containing bovine serum albumin (0.5mg/ml), the slides were incubated with streptavidin-horseradish peroxidase (Vectastain Elite ABC) in PBS containing bovine serum albumin for 90 minutes at room temperature. Detection of biotin-streptavidin-peroxidase complexes was performed by incubating the sections with 0.5mg/ml 3,3' -diaminobenzidine in PBS (pH7.4) and 0.05% H2O 2.

Sections were air dried, fixed with 10% formaldehyde for 10 minutes and washed three times in PBS after permeabilization with 1% triton x-100 for 20 minutes, they were washed three times in PBS, sections were air dried, TUNE L reaction solution was added, sections were incubated at 37 ℃ in a humid box for 1h in the dark, washed three times with PBS, and mounting with DAPI-containing mounting medium.

We found that there was no significant difference in the number of TUNE L + CD31+ cells in the infarct zone of perioperative stroke mice compared to the naive stroke mice, however, there was no significant difference in the number of TUNE L + GFAP + cells (astrocytes) or TUNE L + CD13+ cells (pericytes) in the two groups.

Ten measurement of mouse MIF in plasma

Three days after dMCAO, blood was collected by cardiac puncture under general anesthesia (isoflurane 3%) blood samples were centrifuged at 2000rpm for 10 minutes at room temperature, plasma was collected and frozen at-80 ℃, MIF levels in mouse plasma were quantified using the MIF E L ISA kit (Abcam).

E L ISA demonstrated that 3 days after stroke increased MIF levels in peripheral plasma of perioperative stroke mice compared to stroke alone mice in the experimental group, but no difference was observed 1 day after stroke.

Eleven, flow cytometry

Surgery can induce rapid activation of surrounding immune cells. Among these, mononuclear macrophages are important cell types that recognize tissue damage in the innate immune system. To determine whether perioperative stroke induced macrophages to release MIF, we examined MIF expression by flow analysis.

Single cell suspensions were prepared using RBC lysis Buffer (BD) and filtered through a 70 μm nylon membrane for splenocytes and peripheral blood following the manufacturer's instructions (eBioscience) with anti-mouse CD45, CD11b, F4/80, L y6G, MIF and appropriate isotype controls for staining of single cell suspensions, counting on a FACS Verse cell sorter (BD Biosciences) and using FlowJo software (TreeStar).

We found that the percentage of MIF + F4/80+ cells and the absolute number of MIF + F4/80+ CD11b + CD45+ macrophages in CD45+ CD11b + cells was significantly increased in perioperative stroke 3 days blood and spleen compared to stroke alone mice, but there was no significant difference between the two groups of blood 1 day post-stroke. In addition, tibial fracture surgery alone in mice without stroke may also cause peripheral macrophage MIF expression.

Given that the surgical procedure may be influenced by a variety of factors, such as surgical trauma, stress, pain, etc., we analyzed the potential effects of these factors on MIF expression in animal models by flow cytometry. Surgery, stress, pain and trauma. We found that the percentage of MIF + F4/80+ cells and the MFI of MIF + F4/80+ CD11b + CD45+ macrophages was significantly increased in the operative and trauma groups compared to the control group in CD45+ CD11b + cells. The MFI of the other groups of MIF or MIF + macrophages decreased or remained unchanged. These data indicate that surgical trauma is the major cause of up-regulation of MIF expression in macrophages after surgery and perioperative stroke.

Twelve, MIF antagonist ISO-1 control test

To investigate whether MIF plays a critical role in the post-stroke BBB destruction in perioperative stroke mice, we administered PBS, MIF (60ug/kg) and its antagonist ISO-1(3mg/kg) 3 hours after dMCAO in perioperative and stroke-only mice, we administered MIF or ISO-1 to perioperative and stroke-only mice to investigate the role of MIF in endothelial cell DNA damage and apoptosis, and immunofluorescent staining of TUNE L + CD31+ cells and PARP-1+ CD31+ cells was examined using the following method.

TTC staining shows that 3 days after stroke, exogenous administration of MIF can cause cerebral infarction volume of stroke mice to increase, perioperative stroke mice can have larger cerebral infarction, and ISO-1 can reduce the infarction volume of the perioperative stroke mice. In addition, we also found that 3 days after stroke, BBB destruction was increased in the MIF-treated group, but decreased in the ISO-1-treated group (fig. 2A).

Results it was found that TUNE L + CD31+ cells in the infarct zone were significantly increased in MIF-treated stroke mice and decreased in ISO-1-treated mice (FIG. 2D). We also found that PARP-1+ CD31+ cells were significantly increased in MIF-treated stroke mice and decreased in ISO-1-treated mice.

Thirteen, in vitro blood brain barrier model and oxygen glucose deprivation

To further elucidate the role of MIF in perioperative ischemic stroke-induced BBB injury, we performed OGD experiments on bned 3 cells to establish an in vitro ischemic BBB injury model. In the reoxygenation phase, MIF (100ng/ml) and ISO-1 (30. mu.g/ml) were added to the medium.

bEnd3 cells are maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco BR L) to which 15% fetal bovine serum (FBS, Gibco) is added, in order to simulate in vitro ischemia, the primary culture is replaced with serum-free and glucose-free medium, then the glucose-deficient culture is placed in a modular incubator, which is flushed for 5 minutes with 95% argon and 5% CO2, and then sealed, the chamber is maintained at 37 ℃ for 6 hours, then returned to 95% air, 5% CO2 and glucose-containing medium, during the period specified in each experiment (reoxygenation phase) rMIF and/or ISO-1 are added to the culture.

Immunofluorescent staining results show that OGD/reoxygenation leads to the degradation of zon-1, which can be further exacerbated by MIF treatment (OGD + MIF group), but can be combated by the addition of ISO-1(OGD + M + I group) (fig. 2℃) while OGD/reoxygenation leads to TUNE L + CD31+ cells, which are further exacerbated by MIF treatment and attenuated by the addition of ISO-1.

Fourteen, real time quantitative polymerase chain reaction

To further investigate whether the expression of MIF in endothelial cells was endogenously or peripherally expressed by endothelial cells.

Total RNA from each of the groups of non-OGD, OGD, OGD + MIF and OGD + MIF + ISO-1 was extracted using RNA purification kit (EZ Bioscience). PrimeScript RT kit (Vazyme) was used to synthesize the first part of cDNA with 1. mu.g RNA real-time quantitative polymerase chain reaction was performed using SYBR green PCR Mix (Vazyme) on L ighteCycle 480II (Vazyme). The primers used were β -actin forward primer: 5-GGCTGTATTCCCCTCCATCATCG-3(SEQ ID NO.1), reverse primer: 5-CCAGTTGGTAACAATGCCATGT-3(SEQ ID NO. 2);

MIF forward primer: 5-GCCAGAGGGGTTTCTGTCG-3(SEQ ID NO.3),

reverse primer: 5-GTTCGTGCCGCTAAAAGTCA-3(SEQ ID NO. 4);

PARP-1 forward primer: 5-GGCAGCCTGATGTTGAGGT-3(SEQ ID NO.5), reverse primer: 5-GCGTACTCCGCTAAAAAGTCAC-3(SEQ ID NO. 6);

AIF forward primer: 5-TCCAGAGGCCGAAACAGAG-3(SEQ ID NO.7),

reverse primer: 5-CATTTTGCCCCCTGATGAACC-3(SEQ ID NO. 8);

PUMA forward primer: 5-AGCAGCACTTAGAGTCG-3(SEQ ID NO.9),

reverse primer: 5-CCTGGGTAAGGGGAGGAGT-3(SEQ ID NO. 10).

Expression of GAPDH mRNA was used as an internal control.

qPCR showed no significant change in MIF mRNA in the non-OGD, OGD, OGD + MIF and OGD + MIF + ISO-1 groups. Indicating increased MIF from the periphery.

Fifteen, bEnd3 cell in vitro endocytosis experiment

To elucidate the route of MIF into the b end3 cells, b end3 cells were OGD treated for 6h in the reaeration phase recombinant MIF with or without ISO-1 Dy L light fluorescent label was added to the cell culture.

MIF protein is combined with Dy L light 488NHS esterified substance, bEnd3 cells are marked with CD31 antibody and then combined with Alexa-Fluora-546, the MIF protein marked with fluorescence and the bEnd3 cells are co-cultured for 1-6 hours, washed for a plurality of times by RPMI-1640, then the cells are separated by cell separating liquid, and then the observation is carried out by a fluorescence microscope.

Microscopic analysis showed that MIF was internalized by CD31+ bEnd3 cells. After reoxygenation in CD31+ bEnd3 cells, exogenous MIF was diffusely localized in the cytoplasm and nucleus after 24 hours of incubation. These data indicate that exogenous MIF is endocytosed by the mend 3 cells.

Sixteen, cell transfection

CD74-MIF specific receptors have been reported to be involved in MIF endocytosis. According to our results, CD74 expression was increased in the endothelial cells of PIS mice compared to IS mice. To investigate whether peripheral MIF endocytosis was mediated by the CD74 receptor, we used an in vitro bnd 3 endocytosis model.

In this study, siRNAs were transfected for 24 hours using L ipofectamine 2000(Invitrogen) following the manufacturer's instructions CD74 siRNA (siCD74) (50nM) and siRNA negative control (siNC) (50nM) were purchased from synbiotic (Shanghai, China).

As we show, CD74 was knocked down in the b end3 cells and its knock down efficacy was confirmed by Western blot. The knock-down of CD74 significantly reduced the endocytosis of Dylight-labeled MIF by bEnd3 cells, indicating that the CD74-MIF specific receptor was involved in MIF endocytosis.

Taken together, peripheral surgical trauma induced macrophage activation and up-regulated MIF expression. MIF secreted by macrophages enters endothelial cells through endocytosis mediated by CD74, exerts the activity of endonuclease, and induces single-stranded DNA damage in brain endothelial cells. MIF-mediated endothelial apoptosis is the major mechanism behind PIS to disrupt blood brain barrier integrity and neurological deficits following stroke.

SEQUENCE LISTING

<110> Shanghai university of traffic medical college affiliated renji hospital

Application of macrophage migration inhibitory factor as drug target in treating perioperative stroke

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Claims (5)

1. Application of macrophage migration inhibitory factor as a drug target in treating perioperative stroke.

2. Application of macrophage migration inhibitory factor antagonist in preparing medicine for treating perioperative apoplexy is provided.

3. The use of a macrophage migration inhibitory factor antagonist according to claim 2 in the manufacture of a medicament for the treatment of stroke perioperatively, wherein said macrophage migration inhibitory factor antagonist is ISO-1.

The application of CD74 receptor as a drug target in treating perioperative stroke.

Application of a CD74 receptor blocker in preparing a medicament for treating stroke in perioperative period.

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