阅读说明：本技术 miR-486-3p在制备治疗SAH导致的神经炎症产品中的应用 (Application of miR-486-3p in preparation of product for treating neuroinflammation caused by SAH (neuroinflammation) ) 是由 赖年升 姚阳 李真保 方兴根 吴德刚 袁金龙 赵心同 夏大勇 于 2020-05-20 设计创作，主要内容包括：本发明公开了miR-486-3p在制备治疗SAH导致的神经炎症产品中的应用,本发明提供miR-486-3p相关生物材料的制备如下(a1)或(a2)产品中的应用：(a1)制备治疗蛛网膜下腔出血症的产品；(a2)制备抑制蛛网膜下腔出血导致的神经炎症的产品；所述miR-486-3p的相关生物材料为miR-486-3p,或者装载有miR-486-3p的外泌体,或者能够促进所述miR-486-3p表达的物质。本发明的实验证明用RVG融合到膜糖蛋白Lamp2b修饰的外泌体装载miR-486-3p可以更加有效地将miRNA传递到大脑。miR-486-3p通过抑制Sirtuin2(Sirt2)的表达,发挥抗炎作用。(The invention discloses an application of miR-486-3p in preparation of a product for treating neuroinflammation caused by SAH, and provides an application of miR-486-3p related biomaterial in preparation of a product (a1) or (a2) as follows: (a1) preparing a product for treating subarachnoid hemorrhage; (a2) preparing a product for inhibiting neuroinflammation caused by subarachnoid hemorrhage; the related biological material of the miR-486-3p is miR-486-3p, or an exosome loaded with miR-486-3p, or a substance capable of promoting the expression of miR-486-3 p. Experiments prove that miR-486-3p loaded on exosome modified by RVG fused to membrane glycoprotein Lamp2b can more effectively transfer miRNA to brain. miR-486-3p exerts an anti-inflammatory effect by inhibiting the expression of Sirtuin2(Sirt 2).)

The application of the miR-486-3p related biomaterial in preparing the following products (a1) or (a 2):

(a1) preparing a product for treating subarachnoid hemorrhage;

(a2) preparing a product for inhibiting neuroinflammation caused by subarachnoid hemorrhage;

the related biological material of the miR-486-3p is miR-486-3p, or an exosome loaded with miR-486-3p, or a substance capable of promoting the expression of miR-486-3 p.

2. The use according to claim 1,

the inhibition of neuroinflammation caused by subarachnoid hemorrhage is to play a role in resisting neuritis by inhibiting the expression of Sirt2 through the miR-486-3 p.

3. The use according to claim 1,

the nucleotide sequence of the miR-486-3p is shown in SEQ ID NO. 1.

4. The use according to claim 1,

the exosome loaded with miR-486-3p is an RVG modified exosome.

Use of miR-486-3p or a substance capable of promoting expression of said miR-486-3p in (b1) or (b2) as follows:

(b1) preparing a product for treating subarachnoid hemorrhage;

(b2) preparing a product for inhibiting neuroinflammation caused by subarachnoid hemorrhage.

6. The use according to any one of claims 1 to 5,

the substance capable of promoting the expression of the miR-486-3p is any one of the following substances: a miR-486-3p analog; DNA capable of transcribing to said miR-486-3p, an expression cassette, a recombinant vector or a recombinant cell comprising said DNA.

7. The use according to claim 6,

the nucleotide sequence of the miR-486-3p is shown in SEQ ID NO. 1.

8. The use according to claim 6,

the product is a medicament;

the neuroinflammation resulting from the subarachnoid hemorrhage is caused by Sirt2 mRNA and protein overexpression.

Technical Field

The invention relates to the technical field of biology, in particular to application of miR-486-3p in preparation of a product for treating neuroinflammation caused by SAH.

Background

Aneurysmal subarachnoid hemorrhage (SAH) is usually caused by rupture of the aneurysm, a clinical syndrome with an annual mortality rate of 45% and an incidence of about 6-16 per 10 million, usually young individuals. SAH accounts for 5% -7% of the total incidence of stroke. The major prognostic determinants are Early Brain Injury (EBI) and early Cerebral Vasospasm (CVS) after SAH, and late cerebral ischemia (DCI). More and more studies have shown that EBI after SAH may be a major factor leading to adverse results after SAH. Several pathophysiological processes associated with EBI after SAH are associated with inflammatory cascades. The development of new therapeutic approaches is crucial to promote the recovery of EBI after SAH.

MiRNA (miRNAs) is a family of non-coding RNAs consisting of 17-24 nucleotides that regulate the expression of several target genes at the post-transcriptional level. Previous studies have shown that miRNAs are involved in many physiological/pathological processes and can serve as diagnostic markers and drug targets for a variety of diseases, including stroke, parkinson's disease, Traumatic Brain Injury (TBI), and alzheimer's disease. miRNAs are routinely expressed in cerebrospinal fluid and blood, but changes in miRNAs after SAH are less studied. Moreover, the Blood Brain Barrier (BBB) has been considered to be a major obstacle to drug delivery to the cerebral cortex. It is estimated that 98% of drug molecules are difficult to achieve clinical effects because they cannot cross the BBB.

Exosomes are lipid membrane vesicles of 30 to 150nm diameter, capable of crossing the blood-brain barrier, and can engage in long-range intercellular communication, carrying proteins, lipids, functional mRNA and miRNA to regulate the expression of proteins in target cells. Exosomes released by brain cells are able to cross the blood-brain barrier and can be detected in the blood circulation. Similarly, endothelial cells and perivascular cells also secrete exosomes into the circulation. Exosomes have been used as drug delivery vehicles for the treatment of several central nervous system diseases. Exosomes are also an option for treating stroke. Rabies virus glycoprotein (RVG polypeptide) is a virus component with phagocytosis, can be combined with a nicotine type acetylcholine receptor widely existing on the surfaces of cerebrovascular endothelial cells and nerve cells, and can penetrate through a blood brain barrier, so that the noninvasive cross-blood brain barrier efficient targeted delivery of substances into the brain is realized. The RVG polypeptide can be fused with lysosome-related membrane glycoprotein 2b (LAMP2b) on an exosome membrane, solves the problem of directional transport of exosomes, and can cross BBB and specifically transport miRNAs to a cerebral cortex. In contrast to intraventricular administration, RVG/exosomes (RVG/Exos) are administered intravenously, and are therefore a non-invasive method of treatment of central nervous system disorders. They are capable of rapidly transporting small and large molecule drugs to the central nervous system, while escaping the degradation of the mononuclear phagocyte system.

Disclosure of Invention

The invention aims to provide application of miR-486-3p in preparation of a product for treating neuroinflammation caused by SAH.

The invention provides an application of a miR-486-3p related biomaterial in preparation of a product (a1) or (a 2):

(a1) preparing a product for treating subarachnoid hemorrhage;

(a2) preparing a product for inhibiting neuroinflammation caused by subarachnoid hemorrhage;

the related biological material of the miR-486-3p is miR-486-3p, or an exosome loaded with miR-486-3p, or a substance capable of promoting the expression of miR-486-3 p.

Preferably, the inhibition of neuroinflammation caused by subarachnoid hemorrhage is the effect of inhibiting Sirt2 expression through the miR-486-3p to resist neuritis.

Preferably, the nucleotide sequence of the miR-486-3p is shown in SEQ ID NO. 1.

Preferably, the miR-486-3 p-loaded exosome is an RVG-modified exosome.

The invention also provides application of the miR-486-3p or a substance capable of promoting expression of the miR-486-3p in the following (b1) or (b 2): (b1) preparing a product for treating subarachnoid hemorrhage; (b2) preparing a product for inhibiting neuroinflammation caused by subarachnoid hemorrhage.

Preferably, the substance capable of promoting the expression of miR-486-3p is any one of the following substances: a miR-486-3p analog; DNA capable of transcribing to said miR-486-3p, an expression cassette, a recombinant vector or a recombinant cell comprising said DNA.

Preferably, the nucleotide sequence of the miR-486-3p is shown in SEQ ID NO. 1.

Preferably, the product is a medicament;

the neuroinflammation resulting from the subarachnoid hemorrhage is caused by Sirt2 mRNA and protein overexpression.

The invention has the following advantages:

the experiment of the invention proves that: peripheral injection of modified exosomes (RVG/Exos) loaded miRNAs targeted the hemorrhagic cortex of SAH, thereby modulating its neuroinflammation. Modified exosomes fused to the membrane glycoprotein Lamp2b with RVG may deliver mirnas to the brain more efficiently. miR-486-3p plays an anti-inflammatory role by inhibiting the expression of Sirt 2. The present invention uses RVG/Exos/MiR-486-3p therapy, indicating that intravenous MiR-486-3p loaded RVG/Exos can significantly reduce Sirt2 mRNA and protein levels in brain tissue.

The test of the invention proves that the level of inflammatory cytokines (IL-1 beta, IL-6 and TNF-alpha) in brain tissue is increased after SAH, and the inflammatory cytokines are related to apoptosis (Fluoro-Jade C test), encephaledema and nerve function damage. And RVG/Exos/MiR-486-3p can inhibit the expression of inflammatory cytokines (IL-1 beta, IL-6 and TNF-alpha) in brain tissues after SAH, thereby improving apoptosis, reducing cerebral edema and improving nerve function damage.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a map of the cloning vector pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO provided by the present invention;

FIG. 2 is a fluorescent plot of HBLV-GFP-PURO and HBLV-m-RVG-lamp2b-3xflag-GFP-PURO infection provided by the present invention;

FIG. 3 is a histogram of RVG-LAMP2B overexpression gene expression verified by qPCR provided by the present invention;

FIG. 4 shows the expression levels of miRNA in SAH patients, SAH mouse model plasma exosomes miRNA and SAH mouse model brain tissues provided by the present invention; wherein A is the expression condition of plasma exosome miRNAs of SAH patients; b, miR-486-3p expression in plasma exosomes of SAH mice and Sham mice; miR-486-3p expression in the brains of SAH and Sham mice (24 hours later, n ═ 10);

FIG. 5 is a characteristic representation of exosomes provided by the present invention; wherein, A: expression of Lamp2B in BMSCs and controls after transfection (PC: positive control, HepG 2; BC: blank control, BMSCs; NC: normal control); b: TEM image, scale ═ 200 nm; c: immunoblot analysis of the effects of Lamp2b, CD63, GM130 and ALIX in RVG-Lamp2b modified exosomes and their cells; d: expression levels of miR-486-3p in RVG/Exos/disordered miRNAs (control group) and RVG/Exos/miR-486-3p exosomes were determined by qRT-PCR analysis, with an internal reference of U6 expressed as mean ± SD (. + -. p < 0.001);

FIG. 6 shows the functional verification of the targeted delivery of mouse brain tissue exosomes provided by the present invention; wherein, A: establishing an SAH model and a drug administration strategy; b: immunofluorescence images, i.e. FAM-labeled miRNAs were injected intravenously, unmodified Exos transfected FAM-labeled miRNAs and RVG modified Exos transfected FAM-labeled miRNAs, immunofluorescence images of slices of hemorrhagic cortex (bottom of temporal lobe), the last column being an enlarged image with a scale of 50 μm;

FIG. 7 is a graph of the effect of Exos/miR-486-3p provided by the invention on target gene mRNA expression; wherein, A, B: expression levels 12, 24, 48 and 72h after SAH (n ═ 6); c, D: assessing changes in Sirt2 mRNA and protein expression in Exos/miR-486-3p or Exos/Scr treated mouse brain tissue (n-6); internal parameters were β -actin or p65, expressed as mean ± Sem (SE) (. p <0.05,. p <0.01,. p < 0.001);

FIG. 8 is a graph showing that Exo/miR-486-3p provided by the invention can inhibit apoptosis after SAH and reduce the levels of inflammatory cytokines IL-1 beta, IL-6 and TNF-alpha; wherein, A-C: qRT-P CR assessed changes in IL-1 β, IL-6 and TNF- α expression in the SAH model with an internal parameter of β -actin expression expressed as mean. + -. S E (. SP <0.001, NS: no significant difference; n ═ 6), D-F.ELISA determined the levels of mouse brain tissue inflammatory factors IL-1 β, IL-6 and TNF- α, n ═ 6 (. SP < 0.001);

FIG. 9 is a graph of the effect of Exo/miR-486-3p provided by the invention on neurobehavioral neurological function scores, cerebral edema and neurodegeneration in SAH mice; wherein, A: neurological score of each group of mice (n-18); b: change in brain water content for each group (n ═ 6); c, D: FJC staining and FJC number of positive cells, the first row of the image represents the lower magnification image, while the other images represent the higher magnification images from the first row box, scale 20 μm. All quantitative data were mean ± SE (. about.. p <0.001, NS: no significant difference).

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Animals and ethics, all participants were recruited through the first subsidiary hospital of the southern Anhui medical college, the study followed the Helsinki declaration, and the participants signed informed consent by themselves or by family members. Ethical was passed through the ethical committee of the yies mountain hospital, southern Anhui medical school. All experiments were approved by the ethical committee of the first subsidiary hospital of the southern Anhui medical college and were performed according to the national institutes of health guidelines for animal care and use. All adult male C57BL/6 mice weighing 25-30g were purchased from Shanghai animal center, Chinese academy of sciences, and all were housed in animal rooms at appropriate temperature and humidity, and light/dark for 12 hours each. All efforts were made to minimize the use of animals and to reduce their suffering.

The mouse SAH model of the invention is a mouse pre-circulation SAH model introduced by Mohammed Sabri et al (Stroke. 2011; 42(5): 1454-60.). A male mouse with the weight of 28-32g is anesthetized (4% chloral hydrate is used for anesthesia), then the male mouse is fixed on a stereotaxic apparatus, according to a stereotaxic map of the brain of the mouse, the scalp is cut at the center of the back of the exposed scalp, the skull is exposed, one bone hole is drilled at the position about 4.5mm in front of the front fontanelle, a 27-size spinal puncture needle is slowly inserted into the bone hole at an angle of 40 degrees with the horizontal line for about 4-5mm, the needle touches the skull base and then is withdrawn backwards for 0.5 mm-1 mm, the needle point is ensured to be positioned in the anterior cross pool, then about 50-100 mu l of non-heparinized arterial blood is extracted from the heart of the other mouse and is slowly injected into the anterior cross pool through the spinal puncture needle, and 100 mu. To prevent dehydration of the mice, 1ml of 0.9% physiological saline was injected subcutaneously after the operation. The bottom of the temporal lobe was observed to be frequently stained with blood, mice were sacrificed according to experimental time, and plasma and brain tissue of the mice were retained for subsequent analysis.