CN108714152B

CN108714152B - Application of microRNA98 in preparation of medicine for treating cerebral arterial thrombosis

Info

Publication number: CN108714152B
Application number: CN201810450155.3A
Authority: CN
Inventors: 孙秀兰; 曹露露; 杨进; 郭旭东; 薛腾飞; 季娟; 郭若冰; 黄纪烨
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2018-05-11
Filing date: 2018-05-11
Publication date: 2020-09-18
Anticipated expiration: 2038-05-11
Also published as: CN108714152A

Abstract

The invention discloses an application of microRNA98 in preparation of a medicine for treating cerebral arterial thrombosis, and belongs to the technical field of genetic engineering and medicine research and development. The invention evaluates the effect of microRNA98 in the treatment of ischemic stroke, finds that microRNA98 has a treatment effect on the acute-phase injury of a focal cerebral ischemia-reperfusion rat, and preliminarily clarifies that microRNA98 plays a treatment effect on acute ischemic stroke by inhibiting neuroinflammation in the brain.

Description

Application of microRNA98 in preparation of medicine for treating cerebral arterial thrombosis

Technical Field

The invention belongs to the technical field of genetic engineering and drug research and development, and particularly relates to application of microRNA98 in preparation of a drug for treating cerebral arterial thrombosis.

Background

With the progress of genetic engineering research, scientists have shown a great interest in developing drugs by genetic engineering. MicroRNA is abbreviated as miRNA, usually has the length of 19-25 nucleotides, widely exists in various animals and plants and even single-cell eukaryotes, and is micromolecule single-stranded RNA which is highly conserved in evolution. miRNA generates base complementary pairing with the 3 'non-coding region (3' UTR) of messenger rna (mRNA) of a specific downstream gene (target gene), causing degradation of the mRNA or inhibition of its translation, resulting in failure of protein expression. mirnas are involved in various cellular functions by regulating the expression of target genes. Scientists have demonstrated that the nucleotide sequence of mirnas is highly conserved among different species, with very low mutation rates, with gene clustering, spatio-temporal and tissue specificity of their expression. The miRNA regulates the expression profile of protein at the level after transcription to determine the progress and diversity of a series of important life activities such as cell differentiation, embryonic development and the like, provides a theoretical basis for disclosing a disease generating mechanism from the aspect of RNA regulation and provides a new gene target and a new molecular marker for disease diagnosis and treatment.

In the human genome sequence, microRNA98 is a MicroRNA and is located at Xp11.22. The sequence of microRNA98 was 5'-UGAGGUAGUAAGUUGUAUUGUU-3' (GeneBank: GeneID: 407054). microRNA98 is highly conserved evolutionarily, with homology between different species. Cerebral apoplexy is a nerve function injury caused by abnormal local blood supply to the brain and is divided into two main categories, ischemic stroke and hemorrhagic stroke. Ischemic stroke accounts for about 85% of all stroke cases, and the core mechanism of the damage is excitotoxicity, neuroinflammatory damage and the like caused by cerebral tissue ischemia and hypoxia. Currently, over 500 million people die of stroke every year worldwide; in China, cerebrovascular diseases account for the first causes of death, and with the increasing population of the elderly, stroke patients in China will further increase. Although researchers at home and abroad carry out a great deal of research and progress on the damage and repair mechanism of neurons after ischemia, a practical and effective treatment method is still lacked in clinic except for early thrombolysis. Therefore, the search and development of new therapeutic drugs with exact efficacy are urgent. Whether the microRNA98 is beneficial to treating cerebrovascular diseases such as cerebral arterial thrombosis is not reported at present. The inventor finds that the microRNA98 has a good application prospect in the aspect of treating ischemic stroke.

Disclosure of Invention

The invention aims to solve the technical problem of providing application of microRNA98 in preparing a medicine for treating cerebral arterial thrombosis.

The technical scheme is as follows: the application of the microRNA98 in preparing the medicine for treating the cerebral arterial thrombosis comprises the following sequence of microRNA 98: 5'-UGAGGUAGUAAGUUGUAUUGUU-3' (GeneBank: GeneID: 407054).

Preferably, the administration is performed after the subject has developed symptoms of ischemic stroke.

The cerebral infarction volume of a Middle Cerebral Artery Occlusion (MCAO) model rat can be remarkably reduced and the neurological dysfunction of the rat can be improved by injecting microRNA98 into the lateral ventricle of the rat 24 hours before cerebral ischemia; the micro RNA98 can remarkably reduce the damage of neurons in affected lateral cortex and striatum brain regions of rats in the acute phase of cerebral ischemia reperfusion, inhibit the proliferation and activation of astrocytes and promote the polarization of microglia to M2 type; the microRNA98 is shown to have a protective effect on nerve injury of a focal cerebral ischemia reperfusion rat in an acute phase.

Further research shows that the microRNA98 reduces the expression of inflammatory factors such as IL-1 beta, iNOS and the like in the brain, thereby playing a role in treating ischemic stroke.

Has the advantages that:

the invention evaluates the effect of microRNA98 in the treatment of ischemic stroke, finds that microRNA98 has a treatment effect on the acute-phase injury of a focal cerebral ischemia-reperfusion rat, and preliminarily clarifies that microRNA98 plays a treatment effect on acute ischemic stroke by inhibiting neuroinflammation in the brain.

Drawings

FIG. 1 MicroRNA98 reduces neuromotor dysfunction in rats caused by focal cerebral ischemia-reperfusion nerve injury.

FIG. 2 MicroRNA98 reduced the volume of cerebral infarction caused by focal cerebral ischemia-reperfusion in rats.

FIG. 3 MicroRNA98 reduces neuronal damage caused by focal cerebral ischemia reperfusion in rats.

FIG. 4 MicroRNA98 inhibits astrocyte proliferation and activation due to cerebral ischemia-reperfusion.

Fig. 5 microRNA98 promoted polarization of microglia to M2 type after acute cerebral ischemia-reperfusion.

FIG. 6 MicroRNA98 inhibits the expression level of proinflammatory factors in acute cerebral ischemia-reperfusion injury.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: and (3) preparing a focal cerebral ischemia reperfusion animal model.

A Middle Coronary Artery Occlusion (MCAO) model of the right side of the rat single-filament lumen was prepared using a polylysine-coated nylon wire plug (model: 2634-A4, Beijing Western technologies, Ltd., China) with respect to the Longa method. SD rats are fasted for 8-10 h before operation, the rats are anesthetized by intraperitoneal injection of 10% chloral hydrate (0.4ml/100g, prepared by sterile normal saline), the rats are fixed in a supine position, a median cervical incision is made after iodophor sterilization, the right common carotid artery, the internal carotid artery and the external carotid artery are separated, and a plug thread is inserted from the external carotid artery cut until resistance (about 20mm) appears, which indicates that the plug thread reaches the middle cerebral artery, and the plug thread is ligated. After 90min of ischemia, the thrombus is slowly drawn out, then the external carotid artery is ligated, then the silk thread at the common carotid artery is loosened to recover the blood flow of the right carotid artery, the incision is sutured, then the carotid artery is sterilized by iodophor and placed in a cage, the feeding is carried out at the room temperature of 25 ℃, and the MCAO is finished to finish the focal cerebral ischemia reperfusion model. The whole process uses a Laser Doppler flow meter (MoorFLPI Full-field Laser Perfusion Imager, Moor Instruments Let, UK) to monitor local cerebral blood flow to ensure successful model preparation. From the start of surgery to before the animals were awakened, a thermometer was used to ensure that the body temperature of the rats was maintained at 36.5 ℃. + -. 1 ℃. After anesthesia, only the bifurcation of internal and external carotid arteries was exposed without inserting a wire plug.

Example 2: grouping of experiments and administration.

SD rats were randomly grouped after weighing: sham-surgery group (Sham-Vehicle), cerebral ischemia model group, microRNA98 treatment group.

The administration method comprises the following steps: according to the weight of the rat, 10% chloral hydrate (4.0ml/kg) is injected into the abdominal cavity for anesthesia, and the prone position is fixed, so that the skull is kept horizontal and centered. Shearing hair, sterilizing with iodophor, exposing cranial vertex, and dipping in appropriate amount of H₂O₂The vertex was wiped and bregma was exposed. According to the rat brain atlas, the lateral ventricle coordinates are (AP: 0.8mm, ML: 1.5mm, DV: 4.0mm), the dura mater is drilled according to the coordinate position, the needle is vertically inserted to reach the preset coordinate position, the injection is slowly injected for administration (about 1ul/min), and the needle is left for 10-15 min after the injection is completely finished. Slowly raising the micro sample injection needle back to DV: and (5) reserving a needle at the position of 0.5mm for 2min, taking out the micro sample injection needle, and drilling a hole in a bone wax sealing manner. The rat is taken down from the stereotaxic apparatus, the wound is sutured, and the iodophor is placed back into the rearing cage after being disinfected.

Example 3: a neuromotor function score.

Grading and scoring the neurological deficit of the rat 24h after the operation according to a Bederson method, removing the rat with the neurological deficit of 24h being 0, and calculating a scoring mean value. The criteria are as follows:

0 minute: normal, no neurological symptoms were observed;

1 minute: when the tail is lifted and suspended, the contralateral forelimb of the animal can not be fully extended, and the operation is characterized in that the wrist and elbow are bent, the shoulder is rotated inwards, the elbow is expanded outwards and is tightly attached to the chest wall;

and 2, dividing: placing the animal on a smooth plane, and rotating or circling towards the opposite side of the operation when the animal freely walks;

and 3, dividing: when the animal walks freely, the animal is toppled towards the opposite side of the operation;

and 4, dividing: animals were placed on smooth surfaces without spontaneous walking with a concomitant decrease in the level of consciousness;

and 5, dividing: and death.

In animal experiments, excessive bleeding occurs in the operation process; abnormal breathing, premature death and subarachnoid hemorrhage found at sacrifice after operation are all discarded, and animals removed are randomly supplemented in later experiments. The experimental result is shown in fig. 1, the neuromotor score value given to the microRNA98 treatment group is obviously lower than that given to the model group, and the microRNA98 is shown to be capable of obviously improving the neuromotor dysfunction of rats after cerebral ischemia-reperfusion.

Example 4: determination of cerebral infarction volume.

The rat is subjected to ischemia reperfusion for 24h, and then the brain is taken out by cutting off the head, the brain tissue is rapidly taken out, the olfactory bulb, the cerebellum and the lower brain stem are removed, and the rat is frozen at the temperature of minus 20 ℃ for 15 min. Coronal sections of 2 mm/slice thickness were cut continuously from the anterior pole (electrode pole), incubated in 1% aqueous 2,3,5-triphenyltetrazolium chloride (2,3,5-triphenyltetrazolium chloride, TTC, Sigma, America) in physiological saline at 37 ℃ in the absence of light for 30min, and then fixed in 4% paraformaldehyde solution (pH 7.4) at 4 ℃ overnight. After taking the picture, the cerebral infarct volume was analyzed and calculated using Image J Image analysis software.

As shown in fig. 2, the volume percentage of cerebral infarction of the microRNA 98-treated group was reduced by 40.87% compared to the model group, and the volume of cerebral infarction was significantly reduced.

Example 5: and (5) embedding paraffin and slicing.

The rat is anesthetized by intraperitoneal injection of 10% chloral hydrate solution, a tube is inserted into the left ventricle, physiological saline with the temperature of 37 ℃ is injected rapidly, 4% Paraformaldehyde (PFA) is slowly and uniformly perfused, the brain is taken out after the head is broken, and the brain tissue of the rat is placed in the 4% paraformaldehyde for post-fixation at the temperature of 4 ℃ for 24-48 h. Taking out the fixed brain tissue, trimming, embedding, performing gradient dehydration by ethanol, clearing with xylene, soaking in wax, embedding, cooling the wax block, performing coronal section with paraffin slicer (RM2135, Leica, Germany), collecting forebrain slices with thickness of 5 μm, and taking one at every ten intervals for experiment.

Example 6: and (4) carrying out Nie dyeing.

The slices are dewaxed and hydrated in xylene and alcohol in a gradient manner, tar violet is dripped on the brain slices, the brain slices are incubated for 15min at 37 ℃ in a wet box, and the brain slices are washed twice by distilled water. Placing the slices in color separation liquid, separating color for 5-10s, performing gradient dehydration with alcohol and xylene, sealing, observing Nie corpuscle under the mirror, and taking photograph.

Example 7: immunohistochemical staining.

Drying the slices in a 65 deg.C oven, performing gradient dewaxing hydration with xylene and alcohol, washing with PBS buffer solution for 3 times every 5min, and adding 3% hydrogen peroxide (H)₂O₂) Keeping away from light for 20min to inactivate endogenous peroxideThe enzyme was washed 3 times every 5min in PBS buffer. Soaking the slices in 0.01M (pH6.0) citrate buffer solution heated to boiling by microwave, repairing for 15min at constant temperature by microwave, and naturally cooling to room temperature. After blocking nonspecific antigen with 10% goat serum, primary antibody (antibody concentration shown in Table 1) was added dropwise and incubated overnight at 4 ℃. The following day the primary antibody was washed out with 0.01M PBS 3 times for 5min each time, followed by oven incubation with the secondary antibody added dropwise at 37 deg.C for 1h (antibody concentrations are shown in Table 2), and the secondary antibody was washed out with 0.01M PBS 3 times for 5min each time. Adding Diaminobiphenyl (DAB) (Kaiyu, China) dropwise to the mixture, keeping out of the sun, developing, dehydrating, sealing, observing under a mirror, and taking a photograph.

TABLE 1 Primary antibody for immunohistochemical staining

TABLE 2 Secondary antibodies for immunohistochemical staining

As shown in fig. 3, after 24h of cerebral ischemia reperfusion, compared with the sham operation group, the neuron loss in the model group rats is obvious, the number of neurons in the cortex and striatum is reduced by 27% and 34.9%, respectively, and meanwhile, the number of positive nissl staining is reduced, the staining is lighter and the cell nucleus is shrunk; after the treatment of the microRNA98, the number of neurons in the cortex and the striatum of the rat is remarkably increased compared with that in a model group, and the cell morphology of the neurons is also restored.

As shown in fig. 4, under physiological conditions, astrocyte cell bodies were small, projections were slender, and cell morphology was normal; after 24h of cerebral ischemia reperfusion, the astrocytes of cerebral cortex and striatum of the rat model group are obviously increased, the cell body is enlarged, and the cell protrusion is thickened and shortened. The rat cerebral cortex and striatum of the MicroRNA98 treated group had a reduced number of astrocytes compared to the model group, and the cell morphology returned to normal.

Example 8: and (4) performing fluorescent staining on the immune tissue.

Drying the slices in a 65 ℃ oven, performing gradient dewaxing hydration on xylene and alcohol, dropwise adding 3% hydrogen peroxide prepared by PBS (phosphate buffer solution) and reacting for 15min in a dark place, and washing 3 times every 5min by using PBS buffer solution. Soaking the slices in 0.01M (pH6.0) citrate buffer solution heated to boiling by microwave, repairing for 15min at constant temperature by microwave, and naturally cooling to room temperature. Adding blocking solution (containing 5% goat serum and 0.1% Triton X-100) and blocking at room temperature for 1 hr. Primary antibody (antibody concentration see Table 3) was added dropwise and incubated overnight at 4 ℃. Taking out the mixture the next day, rewarming for 1h, washing with 0.01M PBS for the first antibody for 3 times, 5min each time, adding corresponding fluorescent secondary antibody (antibody concentration shown in Table 4), incubating for 1h at 37 ℃, and washing with 0.01M PBS for 3 times, 5min each time. 5ug/ml hoechest solution was added dropwise and the reaction was protected from light for 15min, and washed with 0.01M PBS. After mounting, observing the expression of the corresponding protein under a fluorescence microscope, and taking a photograph.

TABLE 3 fluorescent staining of immunohistones primary antibody

TABLE 4 immunohistofluorescent staining of secondary antibodies

Under normal physiological conditions, microglia appear as a highly branched resting state with small cells, long or triangular shapes, with elongated branches. Under pathological conditions such as inflammation, infection, trauma or other nervous system diseases in the brain, the activation is rapidly carried out, and the morphologically transformed from a highly quiescent state with multiple branches to an amoeba-like activation state with a large volume, disappearance of processes and rounding of cell bodies. Under the pathophysiological conditions of stroke, microglia exert a biphasic action: on one hand, the microglia polarizes to M1 type, and can play a role in neurotoxicity such as proinflammatory, oxidative stress, damage of blood-brain barrier, nerve regeneration inhibition and the like; on the other hand, microglia polarize to M2 type to exert neuroprotective effects such as anti-inflammation, nourishing nerves, promoting nerve repair, and promoting angiogenesis.

As shown in fig. 5, after 24h of cerebral ischemia-reperfusion, the expression of the M2 type marker TREM2 in the treatment group was significantly increased compared with the expression of the M1 type marker iNOS in the model group, and at the same time, the expression of the M1 type marker iNOS was significantly decreased, which indicates that the administration of microRNA98 for treatment can promote the polarization of brain microglia to M2 type to exert a brain protection effect.

Example 9: western blotting (Western blotting).

Rats were anesthetized by intraperitoneal injection with 10% chloral hydrate solution, sacrificed after decapitation, and brains were rapidly removed on ice, dividing cerebral hemispheres into injured side and non-injured side. Taking fresh brain tissue in the area around the injury side infarction, fully homogenizing the brain tissue and lysate according to the mass-volume ratio of 1:10, putting the homogenate into an ice box, placing on a shaking table for fully lysing for 30min, then centrifuging at 4 ℃ at 12000rpm for 15min, sucking supernatant, adding 6 times of protein loading buffer solution according to the volume ratio, denaturing at 100 ℃ for 15min, subpackaging, and storing at-80 ℃. Protein samples at-80 ℃ were taken out of solution, centrifuged, added to the lanes with a microsyrin, subjected to SDS-polyacrylamide constant pressure gel electrophoresis separation, and transferred to PVDF membrane (Millipore, USA) using an electrotransfer system (miniprotein-III wet transfer unit, Bio-Rad, Hercules, California, USA). Sealing the 10% skimmed milk powder-TBST at room temperature for 1-2 h. Primary antibody (antibody concentration see Table 5) was added and incubated overnight in a shaker at 4 ℃. The next day, the primary antibody was discarded and the membrane washed with TBST for 10min × 4 times, and a secondary antibody labeled with horseradish peroxidase (antibody concentration shown in Table 6) was added thereto, incubated at room temperature for 1h on a shaker, rinsed with TBST (10min × 4), and added with ECL (Pierce, Thermo scientific, USA) as a luminescent substrate to develop the color. Tanon5200 full-automatic chemiluminescence imaging analysis system development analysis. And carrying out semi-quantitative analysis on the ratio of the gray value of the target protein to the gray value of the internal reference beta-actin.

TABLE 5 Primary antibody for Western immunoblotting

TABLE 6 Secondary antibodies for Western immunoblotting

As shown in figure 6, the expression of inflammatory factors iNOS and IL-1 beta in the brain after 24h of cerebral ischemia-reperfusion can be inhibited by the treatment of the microRNA98, which indicates that the inflammatory reaction in the brain after ischemic stroke can be inhibited by the treatment of the microRNA 98.

Sequence listing

<110> Nanjing university of medical science

Application of <120> microRNA98 in preparation of medicine for treating cerebral arterial thrombosis

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>22

<212>RNA

<213> Human (Human)

<400>1

ugagguagua aguuguauug uu 22

Claims

The application of microRNA98 in preparing a medicine for treating cerebral arterial thrombosis is disclosed, wherein the sequence of microRNA98 is shown as SEQID NO. 1.