Pharmaceutical application of aminoacyl-tRNA synthetase 1

Abstract

The invention discloses pharmaceutical application of aminoacyl-tRNA synthetase 1, and aminoacyl-tRNA synthetase 1 participates in attack of aortic dissection by regulating phenotypic transformation of vascular smooth muscle cells. The method comprises the following steps: transfecting human aortic smooth muscle cells by using siAARS1; aARS1 is overexpressed by adeno-associated virus to specifically transfect mouse vascular smooth muscle cells, and aortic dissection formed by BAPN modeling is promoted on the tissue level. According to the invention, siAARS1 is utilized to transfect cells, so that the contraction phenotype of vascular smooth muscle cells is maintained, and the occurrence of aortic dissection can be effectively inhibited.

Description

Technical Field

[0001] The present invention relates to the field of early diagnosis and treatment of aortic dissection disease, and in particular to pharmaceutical use of aminoacyl-tRNA synthetase 1. Background Art

[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.

[0003] Aortic dissection (AD) refers to a tear in the aortic wall caused by various reasons. The circulating blood enters the middle layer of the aortic wall through the tear in the intima, causing a large-scale tear in the middle layer and forming a dissection. AD is a cardiovascular emergency, generally manifested by severe pain, shock and compression symptoms. In recent years, with the improvement of living standards, the incidence rate has tended to rise, and its severity is far higher than cerebral infarction, myocardial infarction and malignant tumors.

[0004] The specific pathogenesis of aortic dissection is unclear. High-risk factors include hypertension, smoking, dyslipidemia, atherosclerosis, genetic factors (Marfan syndrome, Turner syndrome, Noonan syndrome and Ehlers-Danlos syndrome), congenital vascular malformations, idiopathic medial degenerative changes, aortitis, trauma, pregnancy and other factors. Dissection caused by aortic reconstruction or endovascular operation is also common. At present, open surgery or endovascular treatment is mainly selected according to its different classifications in clinical practice. However, due to its complex and changeable clinical manifestations, rapid disease progression, surgical difficulty and high technical requirements, compared with other cardiovascular diseases such as coronary heart disease, the overall treatment level of aortic dissection in my country is still not high. The missed diagnosis rate, hospitalization and preoperative mortality of acute aortic dissection are still high. The overall treatment and clinical research level of aortic dissection in my country needs to be improved.

[0005] Aminoacyl-tRNA synthetase 1 (AARS1) can transfer not only alanine but also serine or glycine with functional consequences. The lactate-binding ability of AARS1 can be explained by the similar molecular structures of alanine and lactate. The lactyltransferase activity of AARS1 suggests that this conserved enzyme has pleiotropic effects. However, the role of AARS1 in cardiovascular disease and its mechanism have not been reported. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide pharmaceutical uses of aminoacyl-tRNA synthetase 1 in view of the deficiencies in the prior art.

[0007] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0008] In a first aspect, the present invention discloses a product for diagnosing aortic dissection disease.

[0009] The product includes a reagent for detecting aminoacyl-tRNA synthetase 1, and the amino acid sequence of the aminoacyl-tRNA synthetase 1 is shown in SEQ ID NO.8.

[0010] Wherein, the product includes a test kit.

[0011] In a second aspect, the present invention discloses the use of aminoacyl-tRNA synthetase 1 in the preparation of a product for diagnosing aortic dissection disease.

[0012] Wherein, the amino acid sequence of the aminoacyl-tRNA synthetase 1 is shown in SEQ ID NO.8.

[0013] Wherein, the product includes a test kit.

[0014] In a third aspect, the present invention discloses the use of siRNA of aminoacyl-tRNA synthetase 1 in the preparation of a drug for treating aortic dissection disease.

[0015] Wherein, the nucleotide sequence of the siRNA for aminoacyl-tRNA synthetase 1 is any one or a combination of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0016] In a fourth aspect, the present invention discloses a drug for treating aortic dissection disease.

[0017] Wherein, the drug comprises siRNA of aminoacyl-tRNA synthetase 1, and the siRNA of aminoacyl-tRNA synthetase 1 is any one of the following: The first SiRNA, the sense strand is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2; The second SiRNA, the sense strand is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4; The third SiRNA, the sense strand is shown in SEQ ID NO.5, and the antisense strand is shown in SEQ ID NO.6.

[0018] In a fifth aspect, the present invention discloses an application of a mouse aortic dissection model in developing or screening products for preventing and / or treating aortic dissection disease.

[0019] The mice were injected with an adeno-associated virus carrying an aminoacyl-tRNA synthetase 1 having a nucleotide sequence as shown in SEQ ID NO.7.

[0020] The mice were injected with aminoacyl-tRNA synthetase 1 adeno-associated virus via tail vein, with a titer of 900-1100 ifu / mouse.

[0021] In a sixth aspect, the present invention discloses an application of a mouse aortic dissection model in developing or screening methods for preventing and / or treating aortic dissection disease.

[0022] The mice were injected with an adeno-associated virus carrying an aminoacyl-tRNA synthetase 1 having a nucleotide sequence as shown in SEQ ID NO.7.

[0023] The mice were injected with aminoacyl-tRNA synthetase 1 adeno-associated virus via tail vein, with a titer of 900-1100 ifu / mouse.

[0024] In the present invention, the siRNA that downregulates the expression level of aminoacyl-tRNA synthetase 1 protein is any one of the following: The first SiRNA, the sense strand is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2; The second SiRNA, the sense strand is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4; The third SiRNA, the sense strand is shown in SEQ ID NO.5, and the antisense strand is shown in SEQ ID NO.6.

[0025] In the present invention, the nucleotide sequence of the lentivirus for upregulating the expression level of aminoacyl-tRNA synthetase 1 protein is shown in SEQ ID NO.7.

[0026] The present invention provides a pharmaceutical use of aminoacyl-tRNA synthetase 1, which participates in the pathogenesis of aortic dissection by regulating the phenotypic transformation of vascular smooth muscle cells. The method comprises transfecting human aortic smooth muscle cells with siAARS1; and specifically transfecting mouse vascular smooth muscle cells with adeno-associated virus overexpressing AARS1, thereby promoting aortic dissection formed by BAPN modeling at the tissue level.

[0027] Beneficial effects:

[0028] The present invention utilizes siAARS1 to transfect cells and maintain the contractile phenotype of vascular smooth muscle cells, thereby effectively inhibiting the occurrence of aortic dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0030] Figure 1Figure 2 is the mRNA expression level of AARS1 in normal control vascular tissue and aortic tissue of patients with aortic dissection. **P<0.01.

[0031] Figure 2 is the protein expression level of AARS1 in normal control vascular tissue and aortic tissue of patients with aortic dissection.

[0032] Figure 3 The expression level of AARS1 mRNA in the aorta of mice fed with water and 0.5% BAPN drinking water: After extracting mRNA from the aorta of mice fed with water and 0.5% BAPN drinking water, the expression of AARS1 was detected by RT-PCR. *P<0.05. **P<0.01.

[0033] Figure 4 The protein expression level of AARS1 in the aorta of mice fed with water and 0.5% BAPN drinking water: After extracting the protein in the aorta of mice fed with water and 0.5% BAPN drinking water, the expression of AARS1 was detected by Western Blot. *P<0.05.

[0034] Figure 5 To detect the expression level of AARS1 after PDGF-BB stimulation of human aortic smooth muscle cells (HASMCs): HASMCs were stimulated with PDGF-BB (20 ng / mL) for 24 hours, and the expression level of AARS1 protein was detected by Western Blot.

[0035] Figure 6 Effect of knocking down AARS1 on contractile phenotype markers of smooth muscle cells: After HASMCs were transfected with AARS1 small interfering RNA, Western Blot was used to detect the expression levels of AARS1 and smooth muscle cell contractile phenotype markers SM-22 and α-SMA proteins. *P<0.05.

[0036] Figure 7 To investigate the effect of overexpression of AARS1 on aortic dissection, control virus and AARS1 virus were injected into the tail vein of mice. Normal drinking water was used as the control group. 0.25% BAPN drinking water was used to construct a mouse aortic dissection model. The aortic vessels were collected 28 days later and gross photographs of the aortic vessels were taken.

[0037] Figure 8 To investigate the effect of viral overexpression of AARS1 on vascular morphology: mice were intraperitoneally injected with control virus and AARS1 virus, normal drinking water was used as the control group, and 0.25% BAPN drinking water was used to construct a mouse aortic dissection model. Aortic vessels were collected after 28 days, and HE staining was used to detect aortic vascular morphology. DETAILED DESCRIPTION

[0038] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0039] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0040] Example 1 1. To explore whether AARS1 is involved in aortic pathology in normal human aortic tissue and aortas of patients with aortic dissection, mRNA was extracted from normal control vascular tissue and aortic tissues of patients with aortic dissection, and the expression of AARS1 was detected by RT-PCR.

[0041] (1) Add 1 mL of Trizol to the sample, pipette thoroughly to lyse, then transfer to an RNase-free centrifuge tube and lyse for 5 minutes.

[0042] (2) Add 200 μL of chloroform to each EP tube, shake vigorously, vortex for a few seconds, and then place on ice for 10 minutes.

[0043] (3) Centrifugal, 4 o C, 12000 rpm, 15 min, carefully aspirate the supernatant (about 500 μL) and transfer it to a new EP tube.

[0044] (4) Add an equal volume of isopropanol, mix by inverting, and let stand at 4°C for 10 min.

[0045] (5) Centrifugal, 4 o C, 12000 rpm, 15 min, discard the supernatant, and wash with 75% ethanol.

[0046] (6) Centrifugal, 4 o C, 12000 rpm, 5 min, discard the supernatant, air-dry, add appropriate amount of DEPC water (20~50μL), and detect RNA concentration.

[0047] (8) The procedure for the RNA reverse transcription reaction system is as follows: (9) Primer design: Primer Premier 5 software was used to assist in primer design. All primers were synthesized by GenScript Biotech Co., Ltd.

[0048] Results: The expression level of AARS1 mRNA was detected by RT-PCR. Compared with the normal aortic tissue, the expression level of AARS1 mRNA in the aortic tissue of patients with aortic dissection was significantly increased ( Figure 1).

[0049] 2. Next, we further verified this result at the protein level. After extracting proteins from normal control vascular tissue and aortic tissue of patients with aortic dissection, we used Western Blot to detect the expression of AARS1.

[0050] (1) SDS-PAGE (polyacrylamide gel) electrophoresis: prepare 12% separation gel and 3% stacking gel. Take 15 μL sample solution and 3 μL 6× loading buffer and mix well. Boil for 5 min to denature the protein and then load the sample. The loading amount per well is about 30 μg. Perform electrophoresis at 110 V constant voltage for about 90 min until bromophenol blue disappears completely.

[0051] (2) Transfer: After electrophoresis, remove the concentrated gel and immerse the gel in protein transfer buffer (3.6 g / L Tris, 300 mL / L methanol, 17.3 g / L glycine) for 10 to 20 min. Transfer the protein bands to the PVDF membrane (SDS-gel at the negative pole and PVDF membrane at the positive pole) by wet transfer, and perform electrophoresis at 0.3 A constant current for 80 min.

[0052] (3) Blocking: After transfer, remove the PVDF membrane, soak it in PBS for 5 min, and then soak it in PBS containing 5% skimmed milk powder (MPBS) for 1 h.

[0053] (4) Primary antibody incubation: After blocking, place the membrane in a hybridization bag, add the antibody, and shake at 4°C overnight.

[0054] (5) Secondary antibody binding: Wash the membrane once with TBS-T (TBS with Tween-20 at a concentration of 0.05%), then wash the membrane 3 times with TBS-T for 10 min. Add horseradish peroxidase-labeled rabbit anti-goat secondary antibody (1% MPBS, 1:2000 dilution) and incubate at room temperature for 1 h. Wash the membrane once with TBS-T, then wash the membrane 3 times with TBS-T for 10 min.

[0055] (6) ECL color development: Mix ECL color development solution A and B before use, add evenly to the membrane surface, expose the membrane in a dark place, and observe the results.

[0056] Results: Compared with normal human aortic tissue, the protein expression level of AARS1 in aortic tissue of patients with aortic dissection was significantly increased ( Figure 2 ).

[0057] 3. To further clarify the expression of AARS1 protein in aortic dissection at the animal level.

[0058] (1) We used 0.5% BAPN drinking water to induce an aortic dissection model and used normal drinking water as a control. Four weeks later, we extracted the aortic vascular tissue of mice and found that compared with the control group, the mRNA expression of AARS1 in the aortic tissue of mice in the BAPN group was significantly higher than that in the control group ( Figure 3 ) and protein ( Figure 4 ) expression levels were significantly increased.

[0059] (2) At the cellular level, we cultured HASMCs and stimulated them with PDGF-BB (20 ng / mL) for 24 hours. Compared with the control group, PDGF-BB stimulation increased the expression level of AARS1 protein ( Figure 5 ).

[0060] The above experimental results suggest that the expression level of AARS1 protein is significantly increased in both cell and animal models of aortic dissection.

[0061] 4. To further clarify the role of AARS1 protein in aortic dissection, we transfected HASMCs cells with small interfering RNA of AARS1.

[0062] (1) Human aortic smooth muscle cells were plated at an appropriate density.

[0063] (2) After the cells were cultured overnight, they were transfected using the liposome method (Lipo 3000).

[0064] (3) Before transfection, wash cells twice with PBS to remove the remaining serum in the culture medium.

[0065] (4) Dissolve the siRNA sequence and liposome Lipo 3000 in serum-free DMEM medium respectively, and gently mix the two liquids within 5 minutes. Let stand at room temperature for 20 minutes. Add dropwise to each well and shake gently. Place the cells under standard culture conditions and replace with fresh normal culture medium overnight to continue culturing.

[0066] (5) Transfection sequence: AARS1 SiRNA sequence: Si-1: SEQ ID NO.1: Sense Strand: 5'-GTGGTAGCTATGTATTTGATT-3'; SEQ ID NO.2: Antisense Strand: 5'-TCAAATACATAGCTACCACTT-3'.

[0067] Si-2: SEQ ID NO.3: Sense Strand: 5'-GGGCTTCTTTGCTACGTTATT-3'; SEQ ID NO.4: Antisense Strand: 5'-TAACGTAGCAAAGAAGCCCTT-3'.

[0068] Si-3: SEQ ID NO.5: Sense Strand: 5'-CAGGCAACATGAAGGATAATT-3'; SEQ ID NO.6: Antisense Strand: 5'-TTATCCTTCATGTTGCCTGTT-3'.

[0069] Results: It was found that after AARS1 protein was knocked down by small interfering RNA, the expression of contractile proteins in HASMCs decreased ( Figure 6 ).

[0070] 5. In order to further verify the role of AARS1 in aortic dissection at the animal level, AARS1 was overexpressed and HE staining was performed.

[0071] 5.1 Overexpression of AARS1 (1) Male C57BL / 6 mice, 10 days old, were divided into groups: Con group: control group, 10-day-old C57BL / 6 mice were injected with control empty adeno-associated virus (titer 1011 ifu / mouse) through the tail vein 2 weeks later, and then given normal saline; AARS1-OE group: AARS1 overexpression group, 10-day-old C57BL / 6 mice were injected with AARS1 overexpression adeno-associated virus (titer 1011 ifu / mouse) through the tail vein 2 weeks later, and then given normal saline; BAPN group: Modeling group, 10-day-old C57BL / 6 mice were injected with control empty adeno-associated virus (titer 1011 ifu / mouse) through the tail vein, and then given 0.25% β-aminopropionitrile (BAPN); BAPN+AARS1-OE group: modeling and AARS1 overexpression group, 10-day-old C57BL / 6 mice were injected with AARS1 overexpression adeno-associated virus (titer 1011 ifu / mouse) through the tail vein, and then given 0.25% β-aminopropionitrile (BAPN); (2) After 4 weeks, aortic vascular tissue was extracted.

[0072] The AARS1 overexpression virus was synthesized by Qingke Gene Medicine Biotechnology Co., Ltd. The AARS1 gene sequence is as follows: SEQ ID NO.7.

[0073] 5.2 HE staining of aorta (1) Place the paraffin sections in an oven at 65°C for 30 min.

[0074] (2) Dewaxing: xylene for 5 min, 3 times. 100% ethanol for 1 min. 95% ethanol for 1 min. 70% ethanol for 1 min.

[0075] (3) Rinse with distilled water until no water droplets remain on the slides, shake off all water on the sections, and stain with hematoxylin for 5 minutes.

[0076] (4) Rinse with tap water to remove floating color on the slices.

[0077] (5) 1% hydrochloric acid ethanol for 1-3 seconds. Wash several times with tap water.

[0078] (6) Scott solution for 1 min, then wash several times with tap water. Shake off all moisture, and stain with eosin for 10 s.

[0079] (7) Wash several times with distilled water to remove the floating color on the slices. Quickly rinse with 70% ethanol. Quickly rinse with 95% ethanol. Quickly rinse with 100% ethanol for 30 seconds, three times in total.

[0080] (8) Xylene for 2 min, 3 times in total. When the xylene is not dry, seal the slide with neutral gum.

[0081] Results: After BAPN modeling for 4 weeks, the aorta of mice was extracted and the gross morphology of the blood vessels was observed. The ascending aorta and aortic arch of the BAPN group mice showed obvious dilatation and hematoma, while the aortic arch of the AARS1-overexpressing mice (AARS1-OE) showed not only obvious dilatation and hematoma, but also dilatation and hematoma of the descending aorta and thoracic aorta, and the severity was higher ( Figure 7 ), HE staining was performed for histological analysis of blood vessels ( Figure 8 ), the results showed that overexpression of AARS1 (AARS1-OE) can promote the occurrence of dissection. HE staining showed that overexpression of AARS1 promoted the formation and range of aortic dissection. The above experimental results can further prove that overexpression of AARS1 protein can promote the occurrence of aortic dissection.

[0082] The above experimental results fully prove that AARS1 is involved in the process of aortic dissection. By transfecting a virus that overexpresses AARS1 and upregulating the protein expression of AARS1, the development of aortic dissection can be promoted. Therefore, we believe that AARS1 protein can be used as a new important target for the clinical treatment of aortic dissection and has potential clinical application value in the prevention and treatment of aortic dissection.

[0083] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A product for diagnosing aortic dissection disease, characterized in that: The invention comprises a reagent for detecting aminoacyl-tRNA synthetase 1, wherein the amino acid sequence of the aminoacyl-tRNA synthetase 1 is shown as SEQ ID NO.

8.

2. The product according to claim 1, characterized in that The products include kits.

3. Use of aminoacyl-tRNA synthetase 1 in the preparation of a product for diagnosing aortic dissection disease, characterized in that: The amino acid sequence of the aminoacyl-tRNA synthetase 1 is shown in SEQ ID NO.

8.

4. The use according to claim 3, characterized in that: The products include kits.

5. Use of siRNA of aminoacyl-tRNA synthetase 1 in the preparation of a drug for treating aortic dissection, characterized in that: The siRNA of aminoacyl-tRNA synthetase 1 is any one of the following: The first SiRNA, the sense strand is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2; The second SiRNA, the sense strand is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4; The third SiRNA, the sense strand is shown in SEQ ID NO.5, and the antisense strand is shown in SEQ ID NO.

6.

6. A drug for treating aortic dissection, characterized in that: It comprises siRNA for aminoacyl-tRNA synthetase 1, wherein the nucleotide sequence of the siRNA for aminoacyl-tRNA synthetase 1 is any one or a combination of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.

3.

7. Use of a mouse aortic dissection model in developing or screening products for preventing and / or treating aortic dissection disease, characterized in that: The mice were injected with an adeno-associated virus expressing the aminoacyl-tRNA synthetase 1 having a nucleotide sequence as shown in SEQ ID NO.

7.

8. The use according to claim 7, characterized in that: The mice were injected with aminoacyl-tRNA synthetase 1 adeno-associated virus via tail vein, with a titer of 900-1100 ifu / mouse.

9. Use of a mouse aortic dissection model in developing or screening a method for preventing and / or treating aortic dissection disease, characterized in that: The mice were injected with an adeno-associated virus expressing the aminoacyl-tRNA synthetase 1 having a nucleotide sequence as shown in SEQ ID NO.

7.

10. The use according to claim 9, characterized in that: The mice were injected with aminoacyl-tRNA synthetase 1 adeno-associated virus via tail vein, with a titer of 900-1100 ifu / mouse.

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