CN115992142B

CN115992142B - Hsa_circ_0000288 related to neuroprotection and application thereof

Info

Publication number: CN115992142B
Application number: CN202211619382.7A
Authority: CN
Inventors: 郭琳; 王允; 高策; 刘志东; 贾晓霞; 嵇建伦; 王清华
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2023-10-13
Anticipated expiration: 2042-12-15
Also published as: CN115992142A

Abstract

The invention relates to biological medicine, in particular to hsa_circ_0000288 related to neuroprotection and application thereof. The sequence of hsa_circ_0000288 is shown in SEQ ID NO. 1. Experiments prove that the brain-targeted exosome of the hsa_circ_0000288 and the entrapped hsa_circ_0000288 have good effects of resisting epileptogenesis and improving cognitive learning capacity and mood disorder accompanied by epileptic mice.

Description

Hsa_circ_0000288 related to neuroprotection and application thereof

Technical Field

The invention relates to biological medicine, in particular to hsa_circ_0000288 related to neuroprotection and application thereof, and particularly relates to hsa_circ_0000288 related to neuroprotection, an exosome and brain targeting exosome of entrapped hsa_circ_0000288 and application thereof.

Background

As a common nervous system disease, epilepsy seriously endangers human health, and causes huge economic and social burden. Epileptic patients often exhibit spontaneous seizures. In addition, patients also exhibit higher order dysfunction such as cognitive dysfunction, impaired learning ability, depression and anxiety.

The antiepileptic drugs used clinically at present are all capable of inhibiting nerve hyperexcitability and controlling epileptic attacks, only controlling convulsion attacks, not improving epileptic progress and also not improving cognitive dysfunction and mood disorder of patients. Therefore, there is a need to develop new antiepileptic drugs.

The circular RNA (circRNA) has a closed circular structure, is not affected by the exonuclease, and can be stably stored in the body for a long time. Compared with other biological molecules (protein, miRNA and the like), the circRNA has better drug-forming property and can be used as a novel nucleic acid drug. However, no viable circRNA drugs have been found in the field of epilepsy treatment.

Disclosure of Invention

In order to solve the technical problems, the invention provides the following technical scheme:

the first object of the present invention is to provide a neuroprotection-related hsa_circ_0000288, the hsa_circ_0000288 having the sequence shown in SEQ ID NO. 1.

A second object of the present invention is to provide an exosome comprising the hsa_circ_0000288.

The third object of the present invention is to provide a method for preparing the exosome, comprising the steps of:

constructing a lentivirus containing the hsa_circ_0000288, and infecting tool cells to obtain a cell line which stably overexpresses the hsa_circ_ 0000288;

after culturing the cell line which stably overexpresses hsa_circ_0000288 for 24-48 hours, the exosomes which encapsulate the hsa_circ_0000288 are centrifuged and collected.

Preferably, the specific procedures of centrifugation and collection of exosomes are: centrifuging the culture medium after culturing for 24-48 h for 8-10 min by 250-320 g, centrifuging the obtained supernatant for 8-10 min by 1800-2500 g, centrifuging the obtained supernatant for 60-80 min by 8000-12000 g, continuously centrifuging the obtained supernatant for 60-80 min by 8000-12000 g repeatedly, collecting the precipitate, re-suspending the precipitate by adopting sterile PBS solution, and collecting the exosomes.

The fourth object of the invention is to provide a brain-targeted exosome, which is prepared by using dioleoyl phosphatidylethanolamine as a coupling agent and jogging rabies virus peptide RVG with surface lipid of the exosome.

Preferably, the amino acid sequence of the rabies virus peptide RVG is shown as SEQ ID NO. 2.

Preferably, the specific operation procedure for the chimeric preparation is: and incubating dioleoyl phosphatidylethanolamine and rabies virus peptide RVG for 1-1.5 h, mixing the obtained product with the exosome according to the mass ratio of 480-520:1, and incubating for 1.5-2.5 h.

The fifth object of the present invention is to provide the use of hsa_circ_0000288 or the exosome or the brain-targeted exosome in the preparation of an antiepileptic drug.

A sixth object of the present invention is to provide an antiepileptic drug comprising at least one of said hsa_circ_0000288, said exosome or said brain-targeted exosome.

Preferably, the medicament further comprises pharmaceutically acceptable excipients or carriers.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides a nerve protection related circRNAhsa_circ_0000288, and after an adeno-associated virus of an overexpression hsa_circ_0000288 is injected into a mouse hippocampus, the hsa_circ_0000288 can obviously improve cognitive disorder and mood disorder of an epileptic mouse, which indicates that the hsa_circ_0000288 can improve epilepsy and related complications and can be used as an antiepileptic research medicament.

2. According to the invention, hsa_circ_0000288 is entrapped in an exosome, and rabies virus peptide RVG is embedded with surface lipid of the exosome to prepare the exosome with brain targeting performance, so that the exosome with brain targeting performance can be used for targeted treatment of epilepsy, and has a better neuroprotective effect; meanwhile, compared with the exosomes derived from mesenchymal stem cells in the existing research, the brain-targeted exosomes are simpler in preparation process and lower in cost.

Drawings

FIG. 1 shows particle size of the entrapped hsa_circ_0000288 exosomes;

FIG. 2 is an inclusion hsa_circ_0000288 exosome characterization; A. surface markers, B, lens electron microscope results;

FIG. 3 is the effect of hsa_circ_0000288 targeted exosomes on the mouse "epileptic ignition model"; A. rate of successful mouse ignition; B. time of successful mouse ignition; in contrast to the group of epileptic models, ^* p<0.05， ^** p<0.01；

FIG. 4 is a mouse seizure score;

FIG. 5 is the effect of hsa_circ_0000288 targeted exosomes on cognitive ability and mood disorders in epileptic mice; A. identification index of new object identification experiment; B. the time of immobility in the mouse tail suspension experiment; ^* p<0.05， ^** p<0.01；

FIG. 6 is the effect of hsa_circ_0000288 targeted exosomes on the residence time of epileptic mice in the open field center; ^* p<0.05， ^** p<0.01；

FIG. 7 is the effect of hsa_circ_0000288 targeting exosomes on epileptic mouse mood disorders; A. in the overhead cross experiment, the mice are in arm opening time; B. in the overhead cross experiment, the number of times the mice enter the open arms; ^* p<0.05， ^** p<0.01。

Detailed Description

The present invention will be described in further detail with reference to specific examples. Those skilled in the art will appreciate that the invention is not limited to these embodiments. Moreover, the present invention may be equivalently replaced, combined, improved, or modified by those skilled in the art in light of the description of the present invention, but are included in the scope of the present invention.

Example 1

Preparation of exosomes entrapped hsa_circ_0000288

1. Preparation of HEK293T cell lines stably expressing hsa_circ_0000288

Using human cell cDNA as a template, hsa_circ_0000288 (the sequence of which is shown in SEQ ID NO. 1) was cloned into the pLv-GFP/Puro plasmid. The transfection reagent was formulated as follows: opti-MEM medium 1ml,pCMV delta R8.22. Mu.g, pCMV-VSV-G0.5. Mu.g, clone pLv-GFP/Puro 1.5. Mu.g of hsa_circ_0000288, xtreme gene 9. Mu.l. Standing at room temperature for 20 min, adding the prepared transfection reagent into HEK293T cell culture solution, continuously culturing for 48 hours, sucking cell culture medium supernatant into a 50mL centrifuge tube, and centrifuging at 4000rpm for 4min at 4 ℃; the precipitate was discarded, and the supernatant was collected and filtered through a 0.45 μm filter. Then carrying out ultra-high speed centrifugation for 2h (4 ℃ C., 20000 rpm), and obtaining white precipitate, namely the amplified virus; the resuspension was carefully blown off with 100 μl sterile phosphate buffer and added dropwise to the cultured HEK293T cells. After 72 hours, puromycin (5. Mu.g/ml) was added for 2 weeks to obtain HEK293T cell line stably overexpressing hsa_circ_0000288.

2. Exosome extraction

HEK293T cells stably overexpressing hsa_circ_0000288 were replaced with exosome-free dulbecco' smodified eagle medium (DMEM) high-sugar medium. After 24-48 h, the culture medium was centrifuged at 300g for 10min, the pellet was removed and the supernatant was collected. Centrifuge 2000g for 10min, collect supernatant, then centrifuge 10000g for 70min, remove pellet and collect supernatant. The supernatant was further centrifuged at 10000g for 70min, and the pellet was collected and resuspended in 100. Mu.l sterile PBS to give exosomes.

3. Exosome RVG markers

RVG (its amino acid sequence is YTIWMPENPRPGTPCDIFTNSRGKRASNG, shown in SEQ ID NO. 2) is combined with the group on the surface of exosome by using dioleoyl phosphatidylethanolamine as a coupling agent. Firstly, distearoyl phosphatidylethanolamine (dioleoylphosphatidylethanolamine N-hydroxyycycline, DOPE-NHS) is incubated with RVG polypeptide for 1 hour to obtain DOPE-RVG, then DOPE-RVG is mixed with the exosomes prepared in the above way according to the mass ratio of 500:1, and incubated for 2 hours at room temperature to obtain the entrapped circ288 brain-targeted exosomes.

4. Exosome identification

Exosomes were identified using surface markers (CD 9, CD63, TSG1, etc.), a nanoparticle size meter, etc., and the morphology of the prepared exosomes was detected using transmission electron microscopy. The exosomes are dissolved in physiological saline at a concentration (referred to as nucleic acid concentration) of 0.2-1 mg/ml. The solvent sources include, but are not limited to, dextrose injection, physiological saline, amino acid injection, lactic acid-ringer's solution, phosphate buffer or sodium dihydrogen phosphate-citrate buffer, and the like.

6. Experimental results

The identification result shows that the prepared exosomes are distributed at about 180-750 nm (figure 1), and the exosome markers such as CD9, CD63, TSG101 and the like are expressed (figure 2A). The prepared surgical body is disc-shaped and annular (figure 2B).

Example 2

Entrapment of human hsa_circ_0000288 brain targeting exosomes can antagonize epileptogenesis

In this example, a model of pentyltotazapine-induced epileptogenesis was selected to evaluate the activity of entrapped humanized hsa_circ_0000288 brain-targeted exosomes against epileptogenesis.

The experimental animals were selected from 30C 57/BL6J mice of 6-8 weeks of age, and randomly divided into model groups (marked as epileptic group) and administration groups (marked as epileptic+circ288), each group being 15. Two groups of mice were subcutaneously injected with 35mg/kg of pentyltogen every two days. Within 60 minutes after each pentylton treatment, animals were observed for appearance of facial tics, nodding, forelimb cramps, general cramps, etc., and scored according to the lambertiy-Kltgaard criteria: 0 minutes, no obvious abnormality exists in animals; score 1, ear or face distortion; 2 minutes, systemic spasmodic wave; 3 minutes, muscle tremors; 4, the limb is deviated to one side and stands; 5 minutes; general stiffness-clonic attacks. The animals were considered to be successfully induced for seizures with three seizure scores exceeding 3 points. The tail vein of the administration group is injected with 0.1mg/ml of the hsa-circ-0000288 brain-targeted exosome physiological saline injection, the administration volume is 0.2 ml/dose, and the injection is carried out once a week for 5 times. The seizure score and animal seizure induction success rate of the two groups of mice were evaluated.

The results indicate that hsa_circ_0000288 can reduce the rate of successful ignition in animals (fig. 3A), extend the time of successful ignition (fig. 3B), reduce the animal epileptic score (fig. 4, only the epileptic score at some time point (multiple of 3) is given for clarity, and sample variance is omitted).

Example 3

Entrapped humanized hsa_circ_0000288 brain targeting exosomes are capable of antagonizing postepileptic cognitive disorders and mood disorders

1. The experimental animal was a C57/BL6J mouse. The epileptic model adopts a mouse model after status epilepticus. Mice were induced for status epilepticus by intraperitoneal injection with pilocarpine (250 mg/kg). After 24 hours, hsa_circ_0000288 brain-targeted exosome treatment was given.

2. Administration method

In this example, 0.1mg/ml of hsa_circ_0000288 brain-targeted exosome saline injection was used. The administration mode is tail vein injection administration, the administration volume is 0.2 ml/dose, and the sign is epilepsia+circ288. The epileptic group was given an equivalent amount of physiological saline. After 7 days, mice were evaluated for cognitive ability and emotional state. Normal mice injected with an equal amount of physiological saline were used as controls.

3. New object identification experiment

The day before testing, animals were pre-conditioned for 5 minutes in the test box. On the day of the experiment, animals first explored two identical objects for 5 minutes. One of the objects is then replaced with a new object. After 15 minutes, the animals explored the two different objects again. The time the animal explores the new and old objects, respectively, is recorded, and the identification index is recorded. The identification index is the percentage of the time the animal explores a new object to the sum of the time it explores the new and old objects.

4. Tail suspension experiment

The tail of the mouse was held with its head hanging downward and its abdomen facing the camera. The activity of the mice was recorded with a camera for 6min and the cumulative immobility time was statistically analyzed.

5. Open field experiment

The mice were placed in a 30X 30cm spontaneous activity box and the total distance of movement of the mice in the box for 5min and the time of activity in the middle area of the box were recorded.

6. Overhead maze test

The mice were placed in the center of the elevated plus maze and the time the mice were on arm and the number of times they were entered on arm were recorded.

7. Data analysis

The experimental results are expressed as mean ± variance, the comparison between groups is by single factor analysis of variance, and the P-value is less than 0.05 to represent the significance of the statistical difference.

8. Experimental results

In the mouse epilepsy model, the new object recognition index of the mouse is reduced (fig. 5A), the immobility time in the tail suspension experiment is increased (fig. 5B), the open field center residence time is reduced (fig. 6), the open arm residence time in the overhead plus maze experiment is reduced (fig. 7A) and the number of times of entering the open arm is reduced (fig. 7B). Compared with the epileptic model mice, the new object identification index of the brain-targeted exosome group given to the entrapped humanized hsa_circ_0000288 is obviously increased (figure 5A), the tail suspension immobility time is obviously reduced (figure 5B), the open field central residence time is obviously increased (figure 6), and the time of opening arms and the times of entering the opening arms in the overhead cross maze experiment are obviously increased (figure 7A). This suggests that entrapment of human hsa_circ_0000288 brain-targeted exosomes can improve cognitive and mood disorders in epileptic mice.

The foregoing disclosure is merely illustrative of some embodiments of the invention, but the embodiments are not limited thereto and variations within the scope of the invention will be apparent to those skilled in the art.

Claims

The use of hsa_circ_0000288, wherein the use of hsa_circ_0000288 in the preparation of an antiepileptic drug, and the sequence of hsa_circ_0000288 is shown in SEQ ID NO. 1.
2. The use according to claim 1, wherein the antiepileptic drug comprises the hsa_circ_0000288 and a pharmaceutically acceptable adjuvant or carrier.