Use of T-5224 in preventing aortic dissection disease in mice

Abstract

The present invention relates to the use of T-5224 in preventing aortic dissection disease in mice. In the present invention, the AP-1 inhibitor T-5224 with a duration of effective concentration for 2 weeks and 3 weeks respectively is used to intervene in mice with an arterial dissection model induced by BAPN. The results show that the aortic dilation rate and rupture rate of the mice are significantly reduced; the survival condition of the mice is significantly improved. The present invention provides a new and effective method for preventing AD, further provides a basis for guiding rational clinical medication, and provides a new idea for the prevention of AD disease clinically.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and specifically to the use of T-5224 in preventing aortic dissection disease in mice. Background Art

[0002] Aortic dissection (AD) is a rare and life-threatening cardiovascular emergency characterized by medial destruction and aortic wall separation. Aortic inflammation is one of the main features of AD. Immune cells infiltrating the media and adventitia can cause increased oxidative stress, expression of inflammatory factors and matrix metalloproteinases (MMPs), thereby promoting apoptosis of vascular smooth muscle cells (VSMCs) and aortic remodeling, playing an important role in the pathogenesis of AD. Risk factors for aortic dissection mainly include hypertension, hereditary arteriopathy, trauma, aortic aneurysm, and various vasculitides.

[0003] AP-1 in mammals mainly consists of a heterodimer mainly composed of c-Jun and c-Fos proteins, which is one of the most critical transcription factors regulating cell growth and differentiation and is involved in important biological processes such as cell proliferation and differentiation. AP-1 can act on the binding site on the promoter of target genes, namely the phorbol ester (12-O-Tetradecano y l p horbol-13-Acetate, TPA) response element, and regulate the transcription of target genes. T-5224 is a selective AP-1 inhibitor with anti-inflammatory activity, which can specifically inhibit the DNA binding activity of c-Fos / c-Jun without affecting the binding activity of other transcription factors. Some studies have shown that T-5224 can inhibit the upregulation of MMPs transcription induced by IL-1β. Summary of the Invention

[0004] Aiming at the defects existing in the prior art, the purpose of the present invention is to provide the use of T-5224 in preventing aortic dissection disease in mice. In the present invention, the AP-1 inhibitor T-5224 with effective concentrations for durations of 2 weeks and 3 weeks respectively is used to intervene in mice with arterial dissection induced by BAPN. The results show that the aortic dilation rate and rupture rate of the mice are significantly reduced; the survival situation of the mice is significantly improved. The above use of T-5224 in preventing aortic dissection disease in mice can further provide a basis for guiding rational clinical medication and provides a new idea for the prevention of aortic dissection disease clinically.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The use of T-5224 in preventing aortic dissection disease in mice, characterized in that T-5224 can be used to prevent aortic dissection disease in mice.

[0007] On the basis of the above-mentioned scheme, T-5224 can reduce the incidence and rupture rate of aortic dissection in mice.

[0008] On the basis of the above-mentioned scheme, T-5224 can reduce the number of breaks in the elastic layer of the aorta and the area of collagen fibers in mice.

[0009] The use of T-5224 according to the present invention in the prevention of aortic dissection disease in mice has the following beneficial effects:

[0010] Through the survival curve and dissection rupture rate of AD model mice, it was found in this experiment that intervention with an effective concentration of T-5224 could delay the occurrence and development of AD over time. Through changes in the mRNA expression level, it was found that T-5224 inhibited the development process of AD by affecting the phenotypic transformation of smooth muscle cells. The present invention provides a new method for preventing aortic dissection and is very likely to become an effective clinical prevention strategy for aortic dissection and rupture. The present invention has good application prospects for the prevention of AD disease clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention has the following drawings:

[0012] Figure 1 For the grouping and drug administration of animal experiments;

[0013] Figure 2 For the survival curve graphs of mice in each group;

[0014] Figure 3 For the bar graphs of the incidence of aortic dissection in mice in each group;

[0015] Figure 4 For the typical ultrasound images of the aorta of mice in each group;

[0016] Figure 5 For the bar graphs of the statistical aorta diameters of mice in each group;

[0017] Figure 6 For the sectional views of Masson staining, EVG staining and Alcien Blue staining of the aorta of mice in each group;

[0018] Figure 7 For the bar graphs of the statistical number of breaks in the elastic lamina and the area of collagen fibers of the aorta of mice in each group;

[0019] Figure 8 For the bar graphs of the statistical gene expressions of contractile (a) and synthetic (b) smooth muscle cells of the aorta of mice in each group. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be further described in detail below with reference to the accompanying drawings.

[0021] 1. Animal experiment: Male SPF-grade C57BL / 6 mice at 3 weeks of age, with a body weight of 10 ± 2 g per mouse. The feeding conditions were a room temperature of 22 ± 2 °C and a 12-h light-dark cycle each.

[0022] (1) Preparation of experimental drugs:

[0023] a. 3-Aminopropionitrile (BAPN): Precisely weigh BAPN powder (Sigma, A3134). According to the body weight of the mice, the intervention in the first week was 2 ml / d of drinking water, and the drinking water was increased by 2 ml per week for the remaining three weeks. It was dissolved in distilled water to a final concentration of 2 mg / kg / dose and used immediately after preparation. g ma, A3134), and according to the body weight of the mice, the intervention in the first week was 2 ml / d of drinking water, and the drinking water was increased by 2 ml per week for the remaining three weeks. It was dissolved in distilled water to a final concentration of 2 y mg / kg / dose and used immediately after preparation. g mg g / kg y / day dose and used immediately after preparation.

[0024] b. T-5224: Precisely weigh T-5224 powder (MCE, HY-12270). According to the body weight of the mice, the gavage volume in the first two weeks of intervention was 50 μl, and the gavage volume in the last two weeks was 100 μl. First, dissolve the powder with 20 μl of DMSO, and then dilute it to a dose of 10 mg / kg in corn oil and store in separate aliquots for use. μ μl, and the gavage volume in the last two weeks was 100 μ μl. First, dissolve the powder with 20 μl of DMSO, and then dilute it to a dose of 10 g mg g / kg in corn oil and store in separate aliquots for use.

[0025] (2) Animal grouping:

[0026] Male SPF-grade C57BL / 6 mice at 3 weeks of age were randomly divided into 4 groups, with 12 mice in each group, namely the control group (Control group), the model group (BAPN group), the T-5224 intervention for two weeks group (BAPN + T-5224 2wk group), and the T-5224 intervention for three weeks group (BAPN + T-5224 3wk group). The Control group was fed with normal water, and the other three groups were fed with BAPN water. The BAPN + T-5224 3wk group was given T-5224 by gavage one week after being fed with BAPN water for 3 weeks; the BAPN + T-5224 2wk group was given T-5224 by gavage two weeks after being fed with BAPN water for 2 weeks. Each mouse in each group was given gavage once a day, and the experimental period was 30 days.

[0027] During the experiment, the mice in each group were allowed to eat and drink freely. The BAPN water and T-5224 were changed twice a week, and the body weight of the mice was measured regularly every week.

[0028] (3) Specimen collection:

[0029] During the experiment, the survival of mice was monitored daily, and the survival curve was recorded. For the mice that died during the experiment, the aorta was dissected and removed on the same day and stored in tissue fixative. For the remaining mice, after the last gavage administration, aortic ultrasound measurement was performed every other day. After recording the aortic diameter, a part of the mouse aorta was dissected and stored in tissue fixative, and a part was snap-frozen in liquid nitrogen and placed in an -80 °C refrigerator for later use.

[0030] 2. Evaluation of pathological indicators

[0031] (1) Preparation of aortic sections:

[0032] Dehydration and infiltration with wax: The aorta was taken out of the tissue fixative, placed in an embedding cassette and labeled. The embedding cassette was placed in a water tank and rinsed with running water for 2 h. After rinsing, the embedding cassettes were neatly arranged and placed in an automatic biological dehydrator. The automatic dehydration program was set, and after 12 h, the dehydration was completed and the cassettes were taken out.

[0033] Tissue embedding: First, the melted paraffin was poured into the mold. The tissue was carefully taken out with heated forceps and quickly placed in the mold with the cross-section facing up. After the paraffin solidified slightly, the base of the embedding cassette was covered, and more wax was added until the base of the embedding cassette was submerged. It was left to cool at room temperature until it solidified.

[0034] Sectioning and spreading: The embedded wax block was taken out of the mold and placed on a biological tissue freezing table. After the wax block cooled, it was fixed on a microtome. First, the section thickness was adjusted for trimming. When a complete lumen appeared, continuous sectioning began, with each section being 5 μm thick. The sections were taken off with forceps and spread flat on the water surface of the spreading machine. The wrinkles were unfolded with curved forceps, and the spreading temperature was 42 °C.

[0035] Retrieving the sections and baking: After the sections were completely spread, they were quickly retrieved with a glass slide, and the excess water was blotted with filter paper. After writing the number label, they were placed on a baking machine and baked at 42 °C for 2 h. After baking, the sections were placed in an oven at 60 - 70 °C overnight to prevent the sections from falling off.

[0036] At this time, the sections can be dewaxed and rehydrated, and the steps are shown in the following table.

[0037]

[0038] (2) Pathological staining

[0039] a. Masson staining:

[0040] ① The sections were stained with the prepared Weigert iron hematoxylin staining solution for 6 min;

[0041] ② Differentiated with acidic ethanol differentiating solution for 10 s and rinsed with water;

[0042] ③ Blued with Masson bluing solution for 5 min and rinsed with distilled water for 1 min;

[0043] ④ Stain with ponceau magenta staining solution for 6 min;

[0044] ⑤ Prepare a weak acid solution according to the ratio of distilled water: weak acid solution = 2:1 in the above operation, and wash with the weak acid working solution for 1 min;

[0045] ⑥ Wash with phosphomolybdic acid solution for 2 min, and wash with the prepared weak acid working solution for 1 min;

[0046] ⑦ Directly put it into aniline blue staining solution and stain for 1 min, and wash with the prepared weak acid working solution for 1 min;

[0047] ⑧ Dehydrate quickly with 95% ethanol for 2 s, dehydrate with absolute ethanol three times, 10 s each time;

[0048] ⑨ Clear with xylene 3 times, 1 min each time, and seal with neutral resin.

[0049] b. EVG staining:

[0050] ① Put the section into the prepared acidic oxidation solution and oxidize for 5 min, and rinse slightly with tap water;

[0051] ② Bleach with acidic bleaching solution for 1.5 min and rinse with tap water for 2 min;

[0052] ③ Rinse slightly with 70% ethanol;

[0053] ④ Stain by covering with aldehyde fuchsin staining solution for 15 min;

[0054] ⑤ Immerse in 70% ethanol and wash 2 times, 30 s each time, until no purple liquid comes out of the section, and rinse slightly with tap water;

[0055] ⑥ Drop-stain with orange G staining solution for 1 - 2 s and rinse slightly with tap water;

[0056] ⑦ Dehydrate with absolute ethanol, dewax and clear with xylene, and seal with neutral resin.

[0057] c. Alcien Blue staining:

[0058] ① Immerse the section in Alcien acidifying solution for 3 min;

[0059] ② Immerse in Alcien staining solution and stain for 45 min, and rinse with running water;

[0060] ③ Counterstain with nuclear fast red staining solution for 7 min and rinse with running water for 1 min;

[0061] ④ Dehydrate with gradient ethanol, clear with xylene, and seal with neutral resin.

[0062] Elastin breaks: The number of elastin breaks in the transverse section of the aorta was counted based on the EVG staining results of aortic sections in each group, and statistical analysis was performed.

[0063] Collagen content: The collagen fiber area in the transverse section of the aorta was counted using ImageJ software based on the Masson staining results of aortic sections in each group, and statistical analysis was performed.

[0064] 3. Detection of the expression levels of genes related to the phenotypic transformation of smooth muscle cells (SMCs)

[0065] Take the intact aorta, add 1 ml of Trizol to the grinding tube, and grind it with a tissue grinder at 8000 rpm for 30 s, 3 times in total. Let it stand on ice for 1 min after each time to extract tissue RNA:

[0066] ① Add 200 μl of chloroform to the ground tube and centrifuge at 12000 r / min for 15 min at 4°C.

[0067] ② Pipette 500 μl of the supernatant into a sterile 1.5 ml EP tube pre-added with an equal volume of pre-cooled isopropanol and let it stand for 10 min.

[0068] ③ Centrifuge at 12000 r / min for 10 min at 4°C.

[0069] ④ Discard the supernatant, add 75% ethanol prepared with pre-cooled DEPC water, and centrifuge at 12000 r / min for 5 min at 4°C.

[0070] ⑤ Repeat step ④.

[0071] ⑥ Discard the supernatant, air-dry at room temperature for 30 min, dissolve the RNA in DEPC water at 4°C, and measure the concentration.

[0072] Perform reverse transcription on the obtained tissue RNA. The amplification system is as follows:

[0073]

[0074] Select ACTB as the internal reference gene, Myh11, Acta2, Myl9, Cnn1, and Ramp1 as the contractile SMC genes, and Col5A1, Col1A1, Cxcl2, and Fn1 as the synthetic SMC genes. The Real time PCR system is as follows:

[0075]

[0076]

[0077] 4. Verification results

[0078] (1) From Figure 2 、 Figure 3 and Figure 4 it can be seen that after the mice with aortic dissection were respectively given the dose of 5 mg / kg / day of T-5224 for 28 days (AP-1 inhibitor) and the dose of 5 mg / kg / day of T-5224 for 21 days (AP-1 inhibitor 2), the survival of the mice with aortic dissection was significantly improved. The rupture rate of the dissection decreased from 54% to 17% and 25%, and the incidence rate decreased from 44% to 33%. Moreover, as time went on, the effect became more significant. Compared with the model group, the aortic diameter of the mice in the T-5224 intervention group decreased, indicating that the aortic dilation also improved significantly.

[0079] (2) As Figure 6 shown, compared with the BAPN group, after the intervention of 5 mg / kg / day of T-5224, the number of broken elastic lamellae in the aorta of the mice decreased, and the area of collagen fibers also decreased.

[0080] (3) Figure 8 The results shown indicate that compared with the BAPN group, after the intervention of T-5224, the expressions of the smooth muscle cell contractility genes Col5A1, Col1A1, Cxcl2, and Fn1 in the mice were down-regulated, indicating enhanced aortic contractile function. The expressions of the smooth muscle cell synthetic genes Myh11, Acta2, Myl9, Ccn1, and Ramp1 were up-regulated, indicating a significant increase in the proliferative ability of smooth muscle cells.

[0081] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those of ordinary skill in the relevant technical field can also make various changes and modifications without departing from the essence and scope of the present invention. Therefore, all equivalent technical solutions also belong to the protection scope of the present invention.

[0082] The content not described in detail in this specification belongs to the well-known prior art of those skilled in the art.

Claims

1. Use of T-5224 in the preparation of a drug for preventing aortic dissection in mice, characterized in that, T-5224 is used for preparing a drug for preventing aortic dissection disease in mice.

2. Use of T-5224 as claimed in claim 1 in the preparation of a medicament for preventing aortic dissection disease in mice, characterized in that: T-5224 can reduce the incidence and rupture rate of aortic dissection in mice.

3. Use of T-5224 as claimed in claim 1 in the preparation of a medicament for preventing aortic dissection disease in mice, characterized in that: T-5224 can reduce the number of broken elastic lamellae in the arteries of mice and reduce the area of collagen fibers.

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