Application of DNASE2 in medicine for preventing and / or treating abdominal aortic aneurysm

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an application of DNASE2 in a medicine for preventing and / or treating abdominal aortic aneurysm, which comprises the following steps: establishing a replicative aging and induced aging model of vascular smooth muscle cells (VSMC) through in-vitro cell culture; the action mechanism of dsDNA excessively accumulated in cytoplasm in abdominal aortic aneurysm (AAA) and the influence of dsDNA scavenging enzyme DNASE2 on senescence of VSMC and AAA are researched, DNASE2 overexpression inhibits formation of AAA of experimental mice, and the application of the dsDNA scavenging enzyme DNASE2 in preparation of drugs with the functions of overexpressing DNASE2 genes or enhancing the scavenging effect of the dsDNA and reducing generation of cytoplasm dsDNA is developed. And new targets and ideas are provided for preventing or treating abdominal aortic aneurysm.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the application of DNASE2 in drugs for preventing and / or treating abdominal aortic aneurysms. Background Art

[0002] Abdominal aortic aneurysm (AAA) is a degenerative lesion characterized by dilation of the abdominal aorta, a common condition in the elderly. Within one year of treatment, 5.3% of AAAs rupture. The mortality rate of a ruptured AAA is nearly 90%. Age is a significant risk factor for AAA development, and AAA screening is a priority for men aged 65 and older, with proven cost-effectiveness.

[0003] Cardiovascular disease is a common problem in the aging global population. The aging of cells within vascular structures is called vascular senescence. Growing evidence supports that vascular senescence contributes to age-related cardiovascular pathology. The proportion of senescent VSMCs in the arterial wall gradually increases, and VSMCs are also susceptible to various stress-induced senescence. Previous studies have found that the expression of the senescence marker P21 mRNA and protein in the aorta tissue of the Ang II-induced ApoE- / - mouse AAA model is significantly higher than that of the control group. However, the specific biological mechanisms by which aging predisposes individuals to AAA are still unclear. Exploring the pathogenesis of AAA based on the characteristics of aging and identifying therapeutic targets for small and medium-diameter AAAs is both scientifically significant and urgent.

[0004] DNA damage response is a key event leading to cellular aging. Damaged DNA fragments in the cell nucleus are mainly released in the form of budding to become double-stranded DNA fragments (dsDNA) in the cytoplasm. Most of them are encapsulated and sequestered in autophagosomes and transferred to lysosomes through fusion for decomposition and clearance.

[0005] Deoxyribonuclease 2 (DNASE2) is a nonspecific deoxyribonuclease that is expressed in most human tissues and preferentially localized in the lysosomes of cells. It can produce single-strand breaks on both strands of dsDNA, thereby clearing cytoplasmic dsDNA. Summary of the Invention

[0006] To address the issues raised in the prior art, the present invention provides the use of DNASE2 in a medicament for preventing and / or treating abdominal aortic aneurysms. Here, the mechanism of action of excessive accumulation of dsDNA in the cytoplasm of VSMCs in AAA and the effect of the dsDNA scavenger enzyme DNASE2 on VSMC aging and AAA were investigated.

[0007] The technical solutions of the present invention are as follows: The present invention provides the use of deoxyribonuclease II in preparing medicine for preventing and / or treating abdominal aortic aneurysm.

[0008] Furthermore, the drug is an expression vector containing deoxyribonuclease II.

[0009] Furthermore, the vector is a lentiviral vector.

[0010] Furthermore, the drug can upregulate the expression level of deoxyribonuclease II protein, enhance the clearance of dsDNA or reduce the generation of cytoplasmic dsDNA.

[0011] Beneficial effects This study investigates the mechanism of action of excessive accumulation of dsDNA in the cytoplasm of vascular smooth muscle cells (VSMCs) in abdominal aortic aneurysm (AAA), as well as the effects of the dsDNA clearance enzyme DNASE2 on VSMC aging and AAA. Overexpression of DNASE2 inhibits the formation of AAA in experimental mice. This provides new targets and ideas for the preparation of drugs that overexpress the DNASE2 gene or enhance the clearance of dsDNA and reduce the production of cytoplasmic dsDNA for the prevention or treatment of abdominal aortic aneurysm. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A is the HE staining image of normal human aorta and AAA tissue, and a bar graph of the number of medial cells per square millimeter; B is the EVG staining image of normal human aorta and AAA tissue, and a bar graph of the elastin ratio.

[0013] Figure 2 The immunofluorescence images of dsDNA in normal human aorta and AAA tissues, and the histogram of fluorescence intensity of individual cells.

[0014] Figure 3 Representative Western blot results of human normal aorta and AAA tissues, and bar graphs showing the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, RIP1, and DNASE2 / γ-H2AX.

[0015] Figure 4 These are the qRT-PCR results of P21, Beclin1, DNASE2, RIP1, and HMGB1 in human normal aorta and AAA tissues.

[0016] Figure 5 The immunohistochemical results of DNASE2 in human normal aorta and AAA tissues, and the bar graph of the immunohistochemical mean values.

[0017] Figure 6 A is a cell culture picture of young and aged HASMC; B is a β-galactosidase staining image and a bar graph of the percentage of blue cells.

[0018] Figure 7 dsDNA immunofluorescence images of young and aged HASMCs and histograms of fluorescence intensity of individual cells.

[0019] Figure 8 The results of western blot analysis of young and aged HASMCs are shown, as well as a bar graph showing the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, RIP1, and DNASE2 / γ-H2AX.

[0020] Figure 9 qRT-PCR results of P21, Beclin1, DNASE2, HMGB1, and RIP1 in young and aged smooth muscle cells.

[0021] Figure 10 A is a cell culture picture of the control group and MOVAS treated with Ara-C; B is a β-galactosidase staining picture of the control group and MOVAS treated with Ara-C, and a bar graph of the percentage of blue cells.

[0022] Figure 11 dsDNA immunofluorescence images of the control group and MOVAS treated with Ara-C, and a histogram of the fluorescence intensity of individual cells.

[0023] Figure 12 The results of MOVAS protein immunoblotting in the control group and after Ara-C treatment, as well as a bar graph showing the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, RIP1, and DNASE2 / γ-H2AX.

[0024] Figure 13 qRT-PCR results of P21, DNASE2, Beclin1, HMGB1, and RIP1 in the control group and MOVAS treated with Ara-C.

[0025] Figure 14 The results of the scratch migration experiment of MOVAS in the control group and after treatment with Ara-C, as well as the bar graph of the migration area at 0h, 12h, 24h, and 48h.

[0026] Figure 15β-galactosidase staining of DNASE2- and DNASE2 lentivirally overexpressed MOVAS (DNASE2+) after treatment with Ara-C, and a bar graph showing the percentage of blue cells.

[0027] Figure 16 The dsDNA immunofluorescence images of DNASE2- and DNASE2 lentivirus-overexpressed MOVAS (DNASE2+) after treatment with Ara-C, and the bar graph of fluorescence intensity of individual cells.

[0028] Figure 17 The western blot results of DNASE2- and DNASE2 lentivirus-overexpressed MOVAS (DNASE2+) are shown, as well as a bar graph showing the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, and RIP1.

[0029] Figure 18 qRT-PCR results of P21, Beclin1, DNASE2, HMGB1, and RIP1 in DNASE2- and DNASE2 lentivirus-overexpressed MOVAS (DNASE2+).

[0030] Figure 19 The figures show the scratch test results of DNASE2- and DNASE2 lentivirus-overexpressed MOVAS (DNASE2+), and the bar graphs of the migration areas at 0h, 12h, 24h, and 48h.

[0031] Figure 20 These are the examination photos of the Sham group and the AAA group on the 0th and 28th days after surgery, and the bar graph of the diameter of the abdominal aorta of mice detected by ultrasound on the 0th, 7th, 14th and 28th days after surgery.

[0032] Figure 21 Images of the abdominal aorta after dissection of mice in the Sham group and AAA group.

[0033] Figure 22 A is the HE staining image of the aorta of mice in the Sham group and AAA group; B is the EVG staining image of the aorta of mice in the Sham group and AAA group; C is the immunofluorescence image of dsDNA in mouse AAA tissue and the bar graph of the immunohistochemistry average value.

[0034] Figure 23 Immunofluorescence images of DNASE2 in the aorta of mice in the Sham group and AAA group, and bar graphs of fluorescence intensity of individual cells.

[0035] Figure 24The results of Western blotting detection of mouse tissues in the Sham group and AAA group, and the bar graph of the relative protein expression levels of γ-H2AX, P21, DNASE2, Beclin1, HMGB1, RIP1, and DNASE2 / γ-H2AX.

[0036] Figure 25 These are the qRT-PCR results of P21, Beclin1, DNASE2, HMGB1, and RIP1 in the tissues of mice in the Sham group and AAA group.

[0037] Figure 26 Fluorescent labeling observation of frozen sections of aorta tissue of mice infected with lentivirus in the control group, DNASE2- group and DNASE2+ group.

[0038] Figure 27 These are the samples of AAA tissues from mice in the DNASE2- and DNASE2+ groups.

[0039] Figure 28 This is a bar graph of the diameter of the abdominal aorta of mice detected by ultrasound on the 0th, 7th, 14th and 28th days after surgery in the DNASE2- group and the DNASE2+ group.

[0040] Figure 29 The bar graph shows the DNASE2 immunohistochemistry results of the aorta of mice infected with lentivirus in the DNASE2- group and DNASE2+ group, as well as the average immunohistochemistry value.

[0041] Figure 30 HE staining images of aortic tissues of AAA mice in the DNASE2- group and DNASE2+ group, and a bar graph of the number of medial cells per square millimeter.

[0042] Figure 31 EVG staining images and elastin ratio bar graphs of AAA aorta tissues of DNASE2- and DNASE2+ mice.

[0043] Figure 32 Immunofluorescence staining images of dsDNA in AAA of mice in the DNASE2- group and DNASE2+ group, and a bar graph of fluorescence intensity of individual cells.

[0044] Figure 33 The western blot results of AAA in DNASE2- and DNASE2+ mice were shown, as well as a bar graph showing the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, and RIP1.

[0045] Figure 34These are the qRT-PCR detection results of P21, Beclin1, DNASE2, HMGB1, and RIP1 in AAA of mice in the DNASE2- group and DNASE2+ group. DETAILED DESCRIPTION

[0046] The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0047] The present invention provides the use of deoxyribonuclease II (DNASE2) in preparing a medicine for preventing and / or treating abdominal aortic aneurysm.

[0048] Preferably, the drug is an expression vector containing DNASE2.

[0049] Preferably, the vector is a lentiviral vector.

[0050] Preferably, the drug can upregulate the expression level of deoxyribonuclease II protein, enhance the clearance of dsDNA or reduce the production of cytoplasmic dsDNA.

[0051] The DNA damage response is a key event in cellular aging. Damaged DNA fragments within the cell nucleus are primarily released through budding into double-stranded DNA fragments in the cytoplasm. Most of these fragments are encapsulated and sequestered within autophagosomes, where they are then transported to lysosomes for degradation and clearance. Deoxyribonuclease 2 (DNASE2) is a nonspecific endodeoxyribonuclease expressed in most human tissues and preferentially localized within lysosomes. It generates single-strand breaks on both strands of dsDNA, thereby clearing cytoplasmic dsDNA.

[0052] This study investigates the mechanism by which excessive accumulation of dsDNA in the cytoplasm of vascular smooth muscle cells (VSMCs) contributes to abdominal aortic aneurysm (AAA) and the impact of the dsDNA scavenger enzyme, DNASE2, on VSMC aging and AAA. DNASE2 overexpression inhibits AAA formation in experimental mice. This finding provides new targets and strategies for the development of drugs that overexpress DNASE2 or enhance dsDNA clearance, thereby reducing cytoplasmic dsDNA production, for the prevention or treatment of AAA.

[0053] Experimental analysis All data in this study were statistically analyzed using GraphPad Prism 8.0 software. Continuous data are presented as mean ± standard error (SEM). Data between the two groups were compared using the t-test for normally distributed data and the rank-sum test for nonnormal data. P < 0.05 indicated statistical significance. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.005, and **** indicates p < 0.0001.

[0054] 1. Expression of cytoplasmic dsDNA and DNASE2 in human abdominal aortic aneurysm tissue Human aorta tissue specimens were divided into normal human aorta group and abdominal aortic aneurysm (AAA) patient aorta group. The abdominal aorta tissues were embedded in paraffin and sliced, and HE staining was performed ( Figure 1 Middle A, 40x microscope view of the medial smooth muscle cell layer (N=5)), EVG staining ( Figure 1 Middle B, 40x microscope view of the medial smooth muscle cell layer (N=5)), immunofluorescence staining ( Figure 2 , blue (DAPI), red (dsDNA), green (α-SMA), 4 fields of view under 40x microscope were randomly selected for each tissue, and 6 tissues in each group (N=6) were detected.

[0055] Figure 1 Figures A and B show that the aortic wall of human AAA tissue is thickened, the outer wall is rough, the structure is disordered, the smooth muscle cells are significantly lost, and the elastic fibers are disordered, detached, and broken. Compared with non-aneurysmal organ donors, the expression level of extranuclear DNA in human AAA tissue is significantly increased (P < 0.05) ( Figure 2 ), which are mainly composed of medial smooth muscle cells and adventitial fibroblasts.

[0056] Subsequently, Western Blot analysis of abdominal aorta tissue revealed that the expression levels of γ-H2AX, P21, Beclin1, HMGB1, RIP1, and DNASE2 proteins in human AAA were significantly increased (P < 0.05) ( Figure 3 ).

[0057] According to the results of qRT-PCR, the mRNA expression levels of P21, Beclin1, HMGB1, RIP1, and DNASE2 in human AAA tissues were also significantly increased (P < 0.05) ( Figure 4 ).

[0058] In addition, the protein ratios of DNASE2 and γ-H2AX in AAA and normal aorta were compared. Figure 5Figure 2. DNASE2 immunohistochemistry results for normal human aorta and AAA tissues. Four randomly selected fields at 40x magnification were averaged for each tissue (N=6). The results showed that the DNASE2 / γ-H2AX ratio in human AAA tissue was lower than that in normal aorta tissue, but the difference was not statistically significant. DNASE2 protein in human AAA tissue was primarily localized in the smooth muscle cells of the media, with minor expression also observed in the fibroblasts of the intima and adventitia. Quantitative analysis revealed significantly elevated DNASE2 expression in the smooth muscle cells of the AAA media (P<0.05).

[0059] 2. Expression of cytoplasmic dsDNA and DNASE2 in a smooth muscle cell replicative senescence model Human aortic smooth muscle cells (HASMC) were purchased from Cell Labs. The cells were cultured in Gibco high-glucose medium supplemented with 10% high-quality fetal bovine serum and 10% penicillin-streptomycin (both anti-inflammatory and anti-fungal). The culture conditions were: 95% air, 5% carbon dioxide, at 37°C, and 70%-80% humidity. Primary human aortic smooth muscle cells (HASMC) were cultured in vitro, and a replicative senescence model of HASMC was established by natural passage.

[0060] Through cell culture, it was observed that HASMC had a typical long spindle shape before the 3rd to 4th generation, and the proliferation rate was fast (average 3-5 days). When cultured to the 12th to 14th generation, the cells gradually became flat and hypertrophic, the cell secretion granules in the cytoplasm increased, the proliferation rate decreased seriously, and even basically entered the growth cycle stagnation (average 15-20 days) ( Figure 6 Middle A).

[0061] By staining for cell senescence β-galactosidase, the data were expressed as 6 random cell fields (N=6) under a 20x microscope, and it was found that the number of positive cells in senescent cells (PD=10-12 generations) was significantly higher than that in young cells (PD=3-4 generations) (P<0.05) ( Figure 6 Middle B).

[0062] By immunofluorescence staining of dsDNA of aging HASMC, blue (DAPI), green (dsDNA), and red (α-SMA), the data were obtained from 12 random cell fields (N=12) under a 40x microscope in each group. The results showed that the dsDNA expression content of aging HASMC was significantly increased compared with young HASMC (P<0.05) ( Figure 7 ).

[0063] Western Blot analysis showed that the expression levels of γ-H2AX, P21, Beclin1, HMGB1, RIP1, and DNASE2 proteins in young HASMCs were significantly higher than those in senescent cells (P < 0.05) ( Figure 8 ).

[0064] At the same time, the qRT-PCR experiments showed that the mRNA expression levels of P21, Beclin1, HMGB1, RIP1, and DNASE2 were significantly higher than those in young cells (P < 0.05) ( Figure 9 ).

[0065] 3. Expression of cytoplasmic dsDNA and DNASE2 in a smooth muscle cell induced senescence model At the same time, the present invention cultured mouse aortic smooth muscle cell line (MOVAS) in vitro, and increased DNA damage in MOVAS by drug stimulation with cytarabine (Ara-C) (20μM, 24 hours), establishing an induced cell senescence model, which was recorded as the Ara-C group. The control group was treated with the same amount of complete cell culture medium. Cell culture showed that the MOVAS cells treated with Ara-C became flat and hypertrophic, and the growth density decreased ( Figure 10 Middle A).

[0066] Cell senescence β-galactosidase staining showed that the number of positive cells in MOVAS treated with Ara-C (10 μM, 24 hours) was significantly higher than that in the control group (P < 0.05) ( Figure 10 Middle B, data are presented from 6 random cell fields at 20x magnification (N=6).

[0067] Compared with the control group, the dsDNA expression level of cells treated with Ara-C was significantly increased (P < 0.05) ( Figure 11 , blue (DAPI), green (dsDNA), red (α-SMA), data are from 12 random cell fields of view in each group at 40x microscope (N=12).

[0068] Similarly, the expression levels of γ-H2AX, P21, Beclin1, HMGB1, RIP1, and DNASE2 proteins in MOVAS treated with Ara-C were significantly higher than those in the control group (P < 0.05) ( Figure 12 ), and the mRNA expression levels of P21, Beclin1, HMGB1, RIP1, and DNASE2 were significantly increased in MOVAS after Ara-C treatment ( Figure 13 The migration ability of MOVAS was significantly decreased after Ara-C treatment (P<0.05) ( Figure 14 ).

[0069] 4. Effect of DNASE2 overexpression on smooth muscle cell senescence Lentiviral infection of mouse aortic smooth muscle cell line (MOVAS): (1) When the MOVAS cell fusion rate is high, trypsin is used to digest the cells, and the number of cells is counted. Finally, 500 μl of cell suspension is added to a 24-well plate, so that the number of cells in the well is about 20,000-40,000. The plate is then cultured in a constant temperature cell incubator. Within 24 hours, cells with high proliferation capacity reach twice their pre-inoculation number.

[0070] (2) The next day, after confirming that the cells are growing well, take out the lentivirus stored at -80°C and thaw it on ice. According to the MOI of MOVAS obtained from the preliminary experiment, which is 75, the amount of virus stock solution required for each well is calculated based on the MOI. The virus stock solution is dissolved in freshly prepared culture medium and added to the well plate in sequence. At the same time, an appropriate volume of infection enhancement solution is added according to the instructions to improve the infection efficiency.

[0071] (3) After the cells and the virus suspension are fully mixed, place the cells in an incubator and incubate for 8-12 hours. Observe their status and find that there is no obvious change compared with the uninfected group. This shows that the lentivirus is not toxic to the cells. Therefore, the culture should be continued and replaced with fresh culture medium after 24 hours.

[0072] (4) Fluorescence microscopy can be used to estimate the infection efficiency of the target cells by chronic viruses. Usually, the intensity of immunofluorescence labeling reaches its peak about 3-4 days after MOVAS infection. During this period, if the cell fusion rate reaches about 80%, the cell number can be subcultured and expanded for subsequent experimental testing.

[0073] (5) If the cell infection efficiency is high, subsequent cell experiments can be carried out directly. If the infection efficiency is not ideal, puromycin is added for screening. Puromycin is added to the culture medium to a final concentration of 10 μg / ml. The medium is replaced after 24 hours of treatment.

[0074] The results showed that the number of positive cells in the DNASE2- group was significantly higher than that in the DNASE2+ group (DNASE2+ group was transfected with lentivirus to overexpress DNASE2, while DNASE2- group was transfected with an empty vector without lentivirus) (P < 0.05) ( Figure 15 Immunofluorescence (IF) showed that the dsDNA expression level in the DNASE2+ group was significantly lower than that in the DNASE2- group (P < 0.05) ( Figure 16 At the same time, DNASE2 was significantly upregulated in the DNASE2+ group, and the protein expression levels of γ-H2AX, P21, Beclin1, HMGB1, and RIP1 were significantly downregulated (P < 0.05) ( Figure 17). qRT-RCR results showed that the mRNA level of DNASE2 in the DNASE2+ group was significantly upregulated, and the mRNA levels of P21, Beclin1, HMGB1, and RIP1 were significantly lower than those in the DNASE2- group (P < 0.05) ( Figure 18 The cell scratch test showed that the cell migration ability of the DNASE2+ group was significantly higher than that of the DNASE2- group ( Figure 19 ).

[0075] 5. Expression of cytoplasmic dsDNA and DNASE2 in experimental mouse AAA model Establishment of Ang II-induced abdominal aortic aneurysm model in ApoE knockout mice: An abdominal aortic aneurysm model was established in ApoE knockout mice by subcutaneous implantation of angiotensin II (Ang II) capsule osmotic pumps. During the modeling period, mice were fed a high-fat diet. Changes in abdominal aortic diameter were assessed by ultrasound on days 0, 7, 14, and 28 after Ang II capsule osmotic pump implantation. Abdominal aortic tissue was harvested on day 28 to confirm successful aneurysm modeling.

[0076] The criterion for judging whether the construction of the mouse abdominal aortic aneurysm model is successful is that the diameter of the abdominal aortic aneurysm wall is 1.5 times larger than the diameter of the adjacent normal aorta wall.

[0077] Preparation of angiotensin II: (1) Filling and installation of the angiotensin II capsule osmotic pump: The Alzet implantable capsule osmotic pump (2004 model) consists of two parts: an irrigator and a flow rate regulator, which are packaged separately. On a sterile operating table, use a sterile syringe and replace the special needle in the osmotic pump package to draw 200 μL of the prepared angiotensin II solution and inject it into the irrigator of the capsule osmotic pump. Then, install the flow rate regulator on the irrigator.

[0078] (2) Activation of angiotensin II implantable capsule osmotic pumps: After all the required angiotensin II capsule osmotic pumps are installed, place the flow rate regulator outlet of the capsule osmotic pump upwards and immerse it in a 50 mL centrifuge tube filled with an appropriate volume of sterile saline. Cover the centrifuge tube lid and use a marker to mark the time and group on the tube wall. Then place the centrifuge tube upright in a 37°C incubator for 48 hours for activation.

[0079] Subcutaneous implantation of capsule osmotic pumps: (1) Place the ApoE mouse in the induction box of the small animal anesthesia machine. After being fully anesthetized with isoflurane, fix the mouse in a prone position on the operating table and maintain anesthesia with low-concentration isoflurane through a mask.

[0080] (2) Shave the hair in the scapular region of the mouse and disinfect the area with 70% alcohol. All surgical instruments are autoclaved. Use ophthalmic scissors to cut the skin and subcutaneous tissue in the scapular region of the mouse horizontally, with an incision length of approximately 1.5 cm. Bluntly dissect the subcutaneous cavity and fully dilate it.

[0081] (3) Use sterile forceps to grasp the activated angiotensin II capsule osmotic pump, with the outlet of the flow rate regulator facing the mouse's buttocks, and insert it into the subcutaneous cavity. Suture the scapular incision with sterile silk suture and disinfect it. While waiting for the mouse to fully wake up, pay attention to keeping the mouse warm. After the mouse wakes up from anesthesia, return it to the cage and feed it normally. The angiotensin II capsule osmotic pump is placed subcutaneously for 28 days.

[0082] Ultrasound detection of abdominal aorta diameter in mice: The diameter of the mouse abdominal aorta was measured using ultrasound. The diameter of the mouse abdominal aorta was measured and recorded using ultrasound at 0, 7, 14, and 28 days after subcutaneous implantation of angiotensin II capsule osmotic pumps. Mice were deprived of food and water for 6 hours prior to ultrasound examination, and their abdominal skin was prepared. After successful intraperitoneal anesthesia, the mice were secured in a supine position on an ultrasound table. An appropriate amount of ultrasound coupling gel was applied to the abdomen. The ultrasound probe was pressed against the abdomen to center the ultrasound image of the mouse abdominal aorta lumen on the display. The ultrasound frequency was adjusted to optimize the image quality. The suprarenal abdominal aorta was located according to the location of the renal arteries. Representative images of the suprarenal abdominal aorta at its maximum diameter were acquired and the data were recorded.

[0083] The experimental results are as follows: The above-mentioned angiotensin II capsule osmotic pump was used to establish an experimental AAA model in mice (referred to as the AAA group, i.e., the abdominal aortic aneurysm model group). The abdominal aorta diameter of the mice was measured by ultrasound on days 0, 7, 14, and 28 after surgery ( Figure 20 ) showed that compared with the Sham group (sham operation group, which also received a capsule osmotic pump but used saline instead of angiotensin II), the abdominal aorta lumen of the AAA group mice was significantly enlarged. At the same time, the abdominal aorta images after mouse dissection showed obvious tumor-like dilation ( Figure 21 ), indicating that the experimental mouse abdominal aortic aneurysm model induced by angiotensin II was successfully established. Figure 22 Figures (A and B) show that compared with the Sham group, the AAA group had arterial wall hypertrophy, structural disorder, smooth muscle cell loss, and significant elastic fiber damage and rupture. Immunohistochemistry showed that the expression of DNASE2 in the smooth muscle cells of the AAA media was significantly higher than that in the Sham group (P < 0.05). Figure 22C), and is mainly distributed in the smooth muscle cells of the media, and a small amount of expression can also be seen in the fibroblasts of the intima and adventitia. Immunofluorescence (IF) detection of dsDNA found that the expression of extranuclear DNA in the AAA group of mice was significantly increased compared with the Sham group (P < 0.05) ( Figure 23 ), mainly in the smooth muscle cell layer of the media. Subsequently, Western blotting results showed that the expression levels of γ-H2AX, P21, Beclin1, HMGB1, RIP1, and DNASE2 proteins in the experimental AAA tissues of mice were significantly higher than those in the Sham group (P < 0.05) ( Figure 24 ). According to the results of qRT-PCR, the mRNA expression levels of P21, Beclin1, HMGB1, RIP1, and DNASE2 in AAA tissues were also significantly increased (P < 0.05) ( Figure 25 ).

[0084] 6. Effect of DNASE2 overexpression on experimental AAA in mice Injection of lentivirus into mouse tail vein: (1) First, remove the mouse from the cage by its tail. Secure it in the quick-release tube of the intravenous injection instrument. Place the tube in the slot on the main unit. Then, place the mouse's tail in the groove under the squeeze plate and turn on the light switch. Next, straighten the mouse's tail and secure it, aligning the blood vessels. Note that the mouse should be in a lateral position at this time, as it has a tail vein on each side. The brightness of the yellow light can be adjusted according to the illumination conditions of the yellow light so that the dark red tail vein is clearly visible.

[0085] (2) Each mouse was injected with 7.6×10 9 For TU-specific lentivirus, dilute the lentivirus to the appropriate titer with 200 μl of sterile saline. Wipe the mouse's tail with an alcohol swab to dilate the blood vessels. Insert the needle from the lower third of the tail, parallel to the tail as much as possible, and gradually inject from the bottom up to avoid leakage from the needle hole. Once the needle tip enters the blood vessel, gently push the syringe handle to feel for resistance. If the needle tip is in the blood vessel, the syringe handle can be pushed smoothly. After the injection, gently compress the needle hole with a cotton ball for 1 minute to prevent bleeding. Then, remove the mouse from the holder and return it to its original housing.

[0086] The experimental results are as follows: The aorta of the mice was infected by DNASE2 overexpression lentivirus injected through the tail vein (denoted as DNASE2+ group, indicating the group injected with lentivirus containing the target gene (viral vector + target gene + marker gene GFP)), and the DNASE2- group indicated the group injected with empty lentivirus without the target gene (containing only vector backbone + marker gene GFP), to ensure that the infection and integration process of the lentivirus and the vector elements (such as promoter, marker gene) would not affect the experimental results; the control group indicated mice of the same strain and age that were not injected with any virus to exclude the influence of the injection operation (such as trauma, stress). Figure 26 The results show that the aorta tissues of DNASE2- and DNASE2+ groups both expressed significant fluorescent marker protein (green). Figure 27 ) and ultrasound measurements of mouse aorta diameter ( Figure 28 ) showed that DNASE2 overexpression significantly reduced the tumorigenicity rate of angiotensin-induced AAA. The DNASE2 expression level in the DNASE2+ group was significantly higher than that in the DNASE2- group (P < 0.05) ( Figure 29 The aortic wall structure of mice in the DNASE2+ group was more complete, and the elastic fibers were less broken, indicating that upregulating the expression level of DNASE2 can slow down the expansion rate of the abdominal aorta diameter in mice and have a certain inhibitory effect on the formation of abdominal aortic aneurysms in mice ( Figure 30 、 31 Immunofluorescence results showed that the dsDNA expression level in the DNASE2- group was significantly higher than that in the DNASE2+ group (P < 0.05) ( Figure 32 ), suggesting that DNASE2 overexpression in mice effectively cleared cytoplasmic dsDNA and enhanced the resistance of the mouse aorta to angiotensin-induced tumor expansion. At the same time, the expression level of DNASE2 in the aorta tissue of the DNASE2+ group of mice was increased, and the expression levels of γ-H2AX, P21, Beclin1, HMGB1, RIP1, and DNASE2 were significantly lower than those in the DNASE2- group (P < 0.05) ( Figure 33 ). qRT-PCR experiments showed that the mRNA level of DNASE2 in the aorta tissue of the DNASE2+ group of mice was significantly higher than that of the DNASE2- group of mice, and the mRNA expression levels of P21, Beclin1, HMGB1, and RIP1 were significantly reduced (P < 0.05) ( Figure 34 ).

[0087] Through these experiments, the present invention investigated the mechanism by which excessive accumulation of dsDNA in the cytoplasm of vascular smooth muscle cells (VSMCs) contributes to abdominal aortic aneurysm (AAA), as well as the impact of the dsDNA scavenger enzyme DNASE2 on VSMC aging and AAA. DNASE2 overexpression inhibited AAA formation in experimental mice. This finding provides new targets and strategies for the development of drugs that overexpress DNASE2 or enhance dsDNA clearance, thereby reducing cytoplasmic dsDNA production, for the prevention or treatment of AAA.

Claims

1. Use of deoxyribonuclease II in the preparation of drugs for preventing and / or treating abdominal aortic aneurysms.

2. The use according to claim 1, characterized in that The drug is an expression vector containing deoxyribonuclease II.

3. The use according to claim 2, characterized in that The vector is a lentiviral vector.

4. The use according to claim 1, characterized in that The drug can increase the expression level of deoxyribonuclease II protein, enhance the clearance of dsDNA or reduce the generation of cytoplasmic dsDNA.

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