Application of DNASE2 in drugs for the prevention and / or treatment of abdominal aortic aneurysms

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of DNASE2 in drugs for the prevention and / or treatment of abdominal aortic aneurysms. A replicative and induced senescence model of vascular smooth muscle cells (VSMCs) was established through in vitro cell culture. The mechanism of action of excessively accumulated dsDNA in the cytoplasm of abdominal aortic aneurysms (AAA) and the effect of the dsDNA-clearing enzyme DNASE2 on VSMC senescence and AAA were studied. Overexpression of DNASE2 inhibited the formation of AAA in experimental mice. This provides new targets and ideas for the preparation of drugs with functions such as overexpression of the DNASE2 gene or enhanced dsDNA clearance and reduced cytoplasmic dsDNA production for the prevention or treatment of abdominal aortic aneurysms.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of DNASE2 in drugs for the prevention and / or treatment of abdominal aortic aneurysms. Background Technology

[0002] Abdominal aortic aneurysm (AAA) is a degenerative disease characterized by dilation of the abdominal aorta, which is prevalent in the elderly. Among patients receiving treatment for AAA, 5.3% will experience rupture within one year. The mortality rate of ruptured AAA is close to 90%. Age is a significant risk factor for AAA, and screening for AAA in men aged 65 and older has proven cost-effective.

[0003] Cardiovascular disease is a prevalent problem due to global population aging. Cellular aging within vascular structures is termed vascular aging, and increasing evidence supports that vascular aging leads to age-related cardiovascular pathological progression. The proportion of aging vascular endothelial cells (VSMCs) in the arterial wall gradually increases, and VSMCs are also susceptible to stress-induced aging. Previous studies found that the mRNA and protein expression of the aging marker p21 in the aortic tissue of an Ang II-induced ApoE- / - mouse AAA model were significantly higher than in the control group. However, the specific biological mechanisms by which aging makes individuals more susceptible to AAA remain unclear. Exploring the pathogenesis of AAA in response to age-related onset and identifying therapeutic targets for small- to medium-diameter AAA is both scientifically significant and urgent.

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

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

[0006] To address the problems raised in the background art, this invention provides the application of DNASE2 in drugs for the prevention and / or treatment of abdominal aortic aneurysms. Here, the mechanism of action of excessively accumulated dsDNA in the cytoplasm of VSMCs in AAA and the effects of the dsDNA clearance enzyme DNASE2 on VSMC senescence and AAA were investigated.

[0007] The technical solution of the present invention is as follows:

[0008] This invention provides the use of deoxyribonuclease II in the preparation of drugs for the prevention and / or treatment of abdominal aortic aneurysms.

[0009] Furthermore, the drug is an expression vector containing deoxyribonuclease II.

[0010] Furthermore, the vector is a lentiviral vector.

[0011] Furthermore, the drug can upregulate the expression level of deoxyribonuclease II protein, enhance the clearance of dsDNA, or reduce the production of cytoplasmic dsDNA.

[0012] Beneficial effects

[0013] This invention investigates the mechanism of action of excessively accumulated dsDNA in the cytoplasm of vascular smooth muscle cells (VSMCs) in abdominal aortic aneurysms (AAA) and the effects of the dsDNA clearance enzyme DNASE2 on VSMC senescence and AAA. Overexpression of DNASE2 inhibited 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 aneurysms. Attached Figure Description

[0014] Figure 1 Image A shows HE staining of a normal human aorta and AAA tissue, and a bar chart of the number of medial cells per square millimeter; Image B shows EVG staining of a normal human aorta and AAA tissue, and a bar chart of the elastin ratio.

[0015] Figure 2 The images show immunofluorescence of dsDNA in normal human aorta and AAA tissue, and bar graphs of fluorescence intensity in single cells.

[0016] Figure 3 This is a bar chart showing representative Western blot results of normal human aorta and AAA tissue, and the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, RIP1, and DNASE2 / γ-H2AX.

[0017] Figure 4 The results of qRT-PCR for P21, Beclin1, DNASE2, RIP1, and HMGB1 in normal human aorta and AAA tissue are shown.

[0018] Figure 5A bar chart showing the DNASE2 immunohistochemical results and average immunohistochemical values ​​of normal human aorta and AAA tissue.

[0019] Figure 6 Image A shows cell culture images of young and aged HASMCs; Image B shows a β-galactosidase staining image and a blue bar chart showing the percentage of cells.

[0020] Figure 7 Immunofluorescence maps of dsDNA in young and aged HASMCs, and bar charts of fluorescence intensity in single cells.

[0021] Figure 8 Bar chart showing the results of Western blot analysis of proteins in young and aged HASMCs, and the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, RIP1, and DNASE2 / γ-H2AX.

[0022] Figure 9 The results of qRT-PCR for P21, Beclin1, DNASE2, HMGB1, and RIP1 in young and aging smooth muscle cells.

[0023] Figure 10 Image A shows cell culture images of the control group and MOVAS treated with Ara-C; Image B shows β-galactosidase staining and blue bar charts of cell percentages of the control group and MOVAS treated with Ara-C.

[0024] Figure 11 The images show the dsDNA immunofluorescence of the control group and MOVAS treated with Ara-C, and the bar chart of fluorescence intensity of a single cell.

[0025] Figure 12 The bar chart shows the results of MOVAS protein immunoblotting in the control group and the Ara-C treated group, as well as the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, RIP1, and DNASE2 / γ-H2AX.

[0026] Figure 13 The results of qRT-PCR for P21, DNASE2, Beclin1, HMGB1, and RIP1 in the control group and MOVAS treated with Ara-C are shown.

[0027] Figure 14 The results of scratch migration experiments on the control group and MOVAS treated with Ara-C are shown in bar charts, along with the migration areas at 0h, 12h, 24h, and 48h.

[0028] Figure 15The image shows a β-galactosidase staining pattern and a blue bar chart of cell percentages for MOVAS (DNASE2+) overexpressing DNASE2- and DNASE2 lentiviruses after Ara-C treatment.

[0029] Figure 16 Immunofluorescence image of dsDNA of MOVAS (DNASE2+) overexpressing DNASE2- and DNASE2 lentiviruses after Ara-C treatment, and bar graph of fluorescence intensity of single cells.

[0030] Figure 17 The bar chart shows the results of Western blot analysis of MOVAS (DNASE2+) overexpressing DNASE2- and DNASE2 lentiviruses, and the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, and RIP1.

[0031] Figure 18 The results of qRT-PCR for P21, Beclin1, DNASE2, HMGB1, and RIP1 in MOVAS (DNASE2+) overexpressed with DNASE2- and DNASE2 lentiviruses are shown.

[0032] Figure 19 The results of scratch assays for MOVAS (DNASE2+) overexpressed with DNASE2- and DNASE2 lentiviruses, and bar charts showing the migration area at 0h, 12h, 24h, and 48h.

[0033] Figure 20 The images show the results of the Sham group and the AAA group on postoperative day 0 and day 28, and the bar charts showing the diameter of the abdominal aorta of mice measured by ultrasound on postoperative day 0, day 7, day 14, and day 28.

[0034] Figure 21 Images of the abdominal aorta after dissection in mice from the Sham and AAA groups.

[0035] Figure 22 Image A shows HE staining of the aorta of mice in the Sham and AAA groups; Image B shows EVG staining of the aorta of mice in the Sham and AAA groups; Image C shows immunofluorescence of dsDNA in mouse AAA tissue and a bar chart of the average immunohistochemical values.

[0036] Figure 23 Immunofluorescence images of DNASE2 in the aorta of mice in the Sham and AAA groups, and bar charts of fluorescence intensity of single cells.

[0037] Figure 24The bar chart shows the results of Western blot analysis of protein tissues from Sham and AAA mice, and the relative protein expression levels of γ-H2AX, P21, DNASE2, Beclin1, HMGB1, RIP1, and DNASE2 / γ-H2AX.

[0038] Figure 25 The results of qRT-PCR for P21, Beclin1, DNASE2, HMGB1, and RIP1 in tissues of mice from the Sham and AAA groups are shown.

[0039] Figure 26 Fluorescent labeling was observed in frozen sections of aortic tissue from mice infected with lentivirus in the control group, DNASE2- group, and DNASE2+ group.

[0040] Figure 27 Images of AAA tissue samples from mice in the DNASE2- and DNASE2+ groups.

[0041] Figure 28 Bar chart showing the diameter of the abdominal aorta in mice detected by ultrasound on days 0, 7, 14, and 28 post-surgery in the DNASE2- and DNASE2+ groups.

[0042] Figure 29 A bar chart showing the DNASE2 immunohistochemical results and average immunohistochemical values ​​of the aorta of mice infected with lentivirus in the DNASE2- and DNASE2+ groups.

[0043] Figure 30 HE staining images of AAA aortic tissue from mice in the DNASE2- and DNASE2+ groups, and bar charts showing the number of medial cells per square millimeter.

[0044] Figure 31 EVG staining images and bar charts showing the elastin ratio in the AAA aortic tissue of mice in the DNASE2- and DNASE2+ groups.

[0045] Figure 32 Immunofluorescence staining images of dsDNA in AAA mice of the DNASE2- and DNASE2+ groups, and bar charts of fluorescence intensity in single cells.

[0046] Figure 33 The bar chart shows the results of Western blot analysis of AAA in mice from the DNASE2- and DNASE2+ groups, as well as the relative protein expression levels of γ-H2AX, P21, Beclin1, DNASE2, HMGB1, and RIP1.

[0047] Figure 34The results of qRT-PCR detection of P21, Beclin1, DNASE2, HMGB1, and RIP1 in AAA mice of the DNASE2- and DNASE2+ groups are shown. Detailed Implementation

[0048] The following examples are intended to illustrate the present invention, and not to further limit the invention.

[0049] This invention provides the use of deoxyribonuclease II (DNASE2) in the preparation of drugs for the prevention and / or treatment of abdominal aortic aneurysms.

[0050] Preferably, the drug is an expression vector containing DNASE2.

[0051] Preferably, the vector is a lentiviral vector.

[0052] Preferably, the drug can upregulate the expression level of deoxyribonuclease II protein, enhance the clearance of dsDNA, or reduce the production of cytoplasmic dsDNA.

[0053] DNA damage is a key event leading to cellular senescence. Damaged DNA fragments within the cell nucleus are released primarily through budding, becoming double-stranded DNA fragments in the cytoplasm. Most of these are encapsulated and isolated within autophagosomes and then transferred to lysosomes for breakdown and clearance via fusion. Deoxyribonuclease 2 (DNASE2) is a non-specific deoxyribonuclease expressed in most human tissues and preferentially localized within lysosomes. It causes single-strand breaks on both strands of dsDNA, thereby clearing cytoplasmic dsDNA.

[0054] This invention investigates the mechanism of action of excessively accumulated dsDNA in the cytoplasm of vascular smooth muscle cells (VSMCs) in abdominal aortic aneurysms (AAA) and the effects of the dsDNA-clearing enzyme DNASE2 on VSMC senescence and AAA. DNASE2 overexpression inhibited AAA formation in experimental mice, providing a new target and approach for the development of drugs that overexpress the DNASE2 gene or enhance dsDNA clearance and reduce cytoplasmic dsDNA production, for the prevention or treatment of abdominal aortic aneurysms.

[0055] Experimental analysis

[0056] All data in this study were statistically analyzed using GraphPad Prism 8.0 software. Continuous variable data are expressed as mean ± standard error (mean ± SEM). For comparisons between two groups, t-tests were used for normally distributed data, and rank-sum tests were used to assess statistical differences for non-normally distributed data. P < 0.05 indicates a statistically significant difference. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.005, and **** indicates p < 0.0001.

[0057] 1. Expression of cytoplasmic dsDNA and DNASE2 in human abdominal aortic aneurysm tissue

[0058] Human aortic tissue specimens were divided into a normal aortic group and an abdominal aortic aneurysm (AAA) patient group. After paraffin embedding and sectioning of the abdominal aortic tissue, HE staining was performed on both groups. Figure 1 Middle A, 40x magnification, media smooth muscle cell layer field (N=5), EVG staining ( Figure 1 Media B, 40x magnification, smooth muscle cell layer of the media (N=5) field of view, immunofluorescence staining ( Figure 2 Blue (DAPI), red (dsDNA), green (α-SMA), four fields of view under a 40x microscope were randomly selected from each tissue, and six tissues were tested in each group (N=6).

[0059] Figure 1 Images A and B show that human AAA tissue exhibits thickened aortic wall, roughened periphery, disordered structure, significant loss of smooth muscle cells, and disordered, detached, and broken elastic fibers. Compared with non-aneurysmal organ donors, the extranuclear DNA expression level in human AAA tissue is significantly increased (P < 0.05). Figure 2 It is mainly composed of the smooth muscle cell layer of the middle membrane and the fibroblasts of the outer membrane.

[0060] Subsequently, Western blot analysis of abdominal aortic tissue revealed significantly elevated expression levels of γ-H2AX, P21, Beclin1, HMGB1, RIP1, and DNASE2 proteins in human aortic AAA (P < 0.05). Figure 3 ).

[0061] According to qRT-PCR results, the mRNA expression levels of P21, Beclin1, HMGB1, RIP1, and DNASE2 were also significantly increased in human AAA tissues (P < 0.05). Figure 4 ).

[0062] In addition, the protein ratio of DNASE2 to γ-H2AX in AAA was compared with that in normal aorta. Figure 5Immunohistochemical results of DNASE2 in normal human aorta and AAA tissues were obtained. Four fields of view under 40x magnification were randomly selected from each tissue and the average value was taken (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 mainly located in the smooth muscle cells of the media, and a small amount of expression was also observed in the fibroblasts of the intima and adventitia. Quantitative analysis showed that the expression level of DNASE2 in the smooth muscle cells of the media of AAA was significantly increased (P<0.05).

[0063] 2. Expression of cytoplasmic dsDNA and DNASE2 in a replicative aging model of smooth muscle cells

[0064] Human aortic smooth muscle cells (HASMCs) were purchased from a cell experimentation company. Cells were cultured in Gibco high-glucose medium containing 10% high-quality fetal bovine serum and 10% penicillin-streptomycin (double antibiotics) under the following incubator conditions: gas phase: air, 95%; carbon dioxide, 5%; temperature: 37℃; humidity: 70%-80%. Primary HASMCs were cultured in vitro, and a replicative cellular senescence model of HASMCs was established through natural passage.

[0065] Cell culture studies showed that HASMCs, before passages 3-4, exhibited a typical elongated spindle shape and rapid proliferation (average 3-5 days). However, upon reaching passages 12-14, the cells gradually became flattened and enlarged, with increased secretory granules in the cytoplasm, a significant decrease in proliferation rate, and even near-complete cell cycle arrest (average 15-20 days). Figure 6 (A)

[0066] Cellular senescence β-galactosidase staining, data presented as 6 random cell fields (N=6) under 20x magnification, revealed a significantly higher number of positive cells in senescent cells (PD=10-12 passages) than in young cells (PD=3-4 passages) (P<0.05). Figure 6 (B)

[0067] Immunofluorescence staining of dsDNA in senescent HASMCs (dsDNA) was performed, with blue (DAPI), green (dsDNA), and red (α-SMA) as the colors. Data were obtained from 12 random cell fields (N=12) under 40x magnification in each group. The results showed that the dsDNA expression level was significantly higher in senescent HASMCs compared to young HASMCs (P<0.05). Figure 7 ).

[0068] 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 ).

[0069] Meanwhile, in qRT-PCR experiments, the mRNA expression levels of P21, Beclin1, HMGB1, RIP1, and DNASE2 were significantly higher than those in young cells (P < 0.05). Figure 9 ).

[0070] 3. Expression of cytoplasmic dsDNA and DNASE2 in a smooth muscle cell-induced aging model

[0071] Simultaneously, this invention cultured the mouse aortic smooth muscle cell line (MOVAS) in vitro and established an induced cell senescence model by stimulating MOVAS with cytarabine (Ara-C) (20 μM, 24 hours) to increase DNA damage. This group was designated as the Ara-C group, while the control group received an equal volume of complete cell culture medium. Cell culture showed that MOVAS cells treated with Ara-C became flattened, hypertrophic, and had a reduced growth density. Figure 10 (A)

[0072] Cellular senescence β-galactosidase staining showed that the number of positive cells in MOVAS treated with Ara-C (10 μM, 24 h) was significantly higher than that in the control group (P < 0.05). Figure 10 In section B, the data are represented as 6 random cell fields of view (N=6) under 20x magnification.

[0073] 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 12 random cell fields of view (N=12) under 40x magnification in each group.

[0074] Similarly, the expression levels of γ-H2AX, P21, Beclin1, HMGB1, RIP1, and DNASE2 proteins in MOVAS cells treated with Ara-C were significantly higher than those in the control group (P < 0.05). Figure 12 Furthermore, the mRNA expression levels of P21, Beclin1, HMGB1, RIP1, and DNASE2 were significantly increased in MOVAS treated with Ara-C. Figure 13 The migration ability of MOVAS was significantly reduced after Ara-C treatment (P < 0.05). Figure 14 ).

[0075] 4. Effects of DNASE2 overexpression on smooth muscle cell senescence

[0076] Lentiviral infection of the mouse aortic smooth muscle cell line (MOVAS):

[0077] (1) When the fusion rate of MOVAS cells is high, trypsin is used to digest the cells, the number of cells is counted, and finally 500 μL of cell suspension is added to a 24-well plate to make the number of cells in the wells about 20,000-40,000. The plate is then placed in a constant temperature cell culture incubator for culture. Within 24 hours, the number of cells with high proliferation capacity reaches twice that before inoculation.

[0078] (2) On the second day, after confirming that the cells are growing well, the lentivirus stored at -80℃ was taken out and thawed on ice. Based on the MOI of MOVAS obtained from the preliminary experiment, which is 75, the amount of virus stock solution required per well was calculated based on the MOI. The virus stock solution was 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 was added according to the instructions to improve the infection efficiency.

[0079] (3) After the cells and virus suspension are fully mixed, the cells are placed in an incubator and incubated for 8-12 hours. The condition is observed and no significant changes are found compared with the uninfected group. This indicates that the lentivirus is not toxic to the cells. Therefore, the culture should continue. After 24 hours, the culture medium is replaced with fresh medium.

[0080] (4) The infection efficiency of chronic viruses on target cells can be estimated by observing with a fluorescence microscope. Usually, the immunofluorescence labeling intensity reaches its peak about 3-4 days after MOVAS infection. If the cell fusion rate reaches about 80% during this period, the cells can be passaged to expand the cell number for subsequent experimental detection.

[0081] (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 make the final concentration 10 μg / ml. The medium is changed after 24 hours of treatment.

[0082] The results showed that the number of positive cells in the DNASE2- group was significantly higher than that in the DNASE2+ group (the DNASE2+ group was transfected with lentivirus to overexpress DNASE2, while the DNASE2- group was transfected with an empty vector without lentivirus) (P < 0.05). Figure 15 Immunofluorescence (IF) showed that the dsDNA expression level was significantly lower in the DNASE2+ group compared with the DNASE2- group (P < 0.05). Figure 16Meanwhile, 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-PCR results showed that the mRNA level of DNASE2 in the DNASE2+ group was significantly upregulated, while the mRNA levels of P21, Beclin1, HMGB1, and RIP1 were significantly lower than those in the DNASE2- group (P < 0.05). Figure 18 Cell scratch assays showed that the cell migration ability of the DNASE2+ group was significantly higher than that of the DNASE2- group. Figure 19 ).

[0083] 5. Expression of cytoplasmic dsDNA and DNASE2 in the experimental mouse AAA model

[0084] Establishment of an Ang II-induced ApoE gene knockout mouse model of abdominal aortic aneurysm:

[0085] An angiotensin II (Ang II) capsule osmotic pump was subcutaneously implanted into ApoE gene knockout mice to establish a mouse abdominal aortic aneurysm model. The mice were fed a high-fat diet during the modeling period. Changes in the diameter of the abdominal aorta were measured by ultrasound on days 0, 7, 14, and 28 after subcutaneous implantation of the angiotensin II capsule osmotic pump. Abdominal aortic tissue was harvested on day 28 to determine whether the aneurysm model was successfully established.

[0086] The criterion for judging whether the mouse abdominal aortic aneurysm model is successfully constructed is: the diameter of the abdominal aortic aneurysm wall is 1.5 times larger than the diameter of the adjacent normal aortic wall.

[0087] Preparation of angiotensin II:

[0088] (1) Filling and installation of the angiotensin II capsule osmotic pump: The Alzet implantable capsule osmotic pump (model 2004) consists of two parts: an infusion device and a flow rate regulator, which are packaged separately. On a sterile operating table, using a sterile syringe and replacing the special needle in the osmotic pump package, draw 200 μL of the prepared angiotensin II solution and inject it into the infusion device of the capsule osmotic pump. Then, install the flow rate regulator onto the infusion device.

[0089] (2) Activation of angiotensin II implantable capsule osmotic pump: After all the required angiotensin II capsule osmotic pumps are installed, place the flow rate regulator outlet of the capsule osmotic pump with the outlet facing upwards, immerse it in a 50mL centrifuge tube containing an appropriate volume of sterile physiological saline, cover the centrifuge tube, and mark the time and grouping on the tube wall with a marker. Then, place the centrifuge tube vertically in a 37℃ constant temperature incubator for 48 hours to activate it.

[0090] Subcutaneous implantation of capsule osmotic pumps:

[0091] (1) Place the ApoE mice in the induction box of the small animal anesthesia machine. After being fully anesthetized with isoflurane, fix the mice in a prone position on the operating table and maintain the anesthesia state with a low concentration of isoflurane through a mask.

[0092] (2) Shave the hair in the scapular region of the mouse and disinfect the area with 70% alcohol. All surgical instruments were autoclaved. Use ophthalmic scissors to make a transverse incision of the skin and subcutaneous tissue in the scapular region of the mouse, with an incision length of about 1.5 cm. Bluntly dissect and fully dilate the subcutaneous cavity.

[0093] (3) Using sterile forceps, grasp the activated angiotensin II capsule osmotic pump, ensuring the flow regulator outlet faces the mouse's buttocks, and insert it into the subcutaneous cavity. Suture the scapular region incision with sterile silk sutures and disinfect. Keep the mouse warm while waiting for it to fully recover from anesthesia. After recovery, return the mouse to its natural cage for normal care. The angiotensin II capsule osmotic pump should be placed subcutaneously for 28 days.

[0094] Ultrasound measurement of the diameter of the abdominal aorta in mice:

[0095] The diameter of the abdominal aorta in mice was measured using ultrasound. The diameter of the abdominal aorta in mice was measured and recorded by ultrasound on days 0, 7, 14, and 28 after subcutaneous implantation of angiotensin II capsule osmotic pumps. Mice were deprived of water and food for 6 hours before ultrasound examination, and the abdominal skin was prepared. Mice were successfully anesthetized via intraperitoneal injection and fixed in a supine position on the ultrasound examination 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 abdominal aorta on the display screen, and the ultrasound frequency was adjusted to achieve optimal image quality. The suprenal abdominal aorta was located based on the position of the renal artery. Representative images of the largest diameter of the suprenal abdominal aorta were acquired, and the data were recorded.

[0096] The experimental results are as follows:

[0097] An experimental AAA model was established in mice using the angiotensin II capsule osmotic pump method (referred to as the AAA group, i.e., the abdominal aortic aneurysm model group). The diameter of the abdominal aorta in mice was measured by ultrasound on postoperative days 0, 7, 14, and 28. Figure 20 The results showed that, compared with the Sham group (sham surgery group, i.e., the same subcutaneous implantation of a capsule osmotic pump but with saline replacing angiotensin II), the abdominal aorta lumen of mice in the AAA group was significantly enlarged. Simultaneously, images of the abdominal aorta after dissection of the mice showed obvious aneurysmal dilatation of the abdominal aorta. Figure 21 This indicates that the angiotensin II-induced experimental mouse abdominal aortic aneurysm model has been successfully established. The results were confirmed by HE staining and elastic fiber protein staining. Figure 22As shown in Figures A and B, compared with the Sham group, the AAA group exhibited significant arterial wall hypertrophy, structural disorder, smooth muscle cell loss, and marked elastic fiber damage and breakage. Immunohistochemistry revealed that the expression level of DNASE2 in the smooth muscle cells of the AAA group was significantly higher than that in the Sham group (P < 0.05). Figure 22 It is mainly expressed in the medial smooth muscle cells, with small amounts also observed in the intima and adventitia fibroblasts. Immunofluorescence (IF) detection of dsDNA revealed a significant increase in extranuclear DNA expression in the AAA mouse group compared to the Sham group (P < 0.05). Figure 23 The primary cell layer was the tunica media smooth muscle cell layer. Subsequently, Western blot results showed that the expression levels of γ-H2AX, P21, Beclin1, HMGB1, RIP1, and DNASE2 proteins in the mouse experimental AAA tissue were significantly higher than those in the Sham group (P < 0.05). Figure 24 According to qRT-PCR results, the mRNA expression levels of P21, Beclin1, HMGB1, RIP1, and DNASE2 were also significantly increased in AAA tissues (P < 0.05). Figure 25 ).

[0098] 6. Effects of DNASE2 overexpression on AAA in experimental mice

[0099] Lentivirus injected into the tail vein of mice:

[0100] (1) First, remove the mouse by its tail and take it out of the cage. Fix it in the quick-release mouse tube of the intravenous injection instrument, place the mouse tube in the slot on the main unit, and then place the mouse tail in the groove under the squeezing plate. Turn on the light switch. Next, straighten and fix the mouse's tail, and position the blood vessel correctly. Note that the mouse should be in a lateral recumbent position at this time, because there is a tail vein on each side. The brightness of the yellow light can be adjusted according to the illumination of the yellow light to make the dark red tail veins clearly visible.

[0101] (2) Each mouse was injected with 7.6 × 10 9 For TU lentivirus, dilute the lentivirus to an appropriate titer with 200 μL of sterile saline. Wipe the mouse's tail with an alcohol swab to dilate blood vessels. Insert the needle into the lower third of the tail at an angle parallel to the tail, gradually increasing the injection depth to avoid leakage from previous injection sites. Once the needle is in the blood vessel, gently push the syringe handle to feel the resistance; if the needle is in the blood vessel, the syringe handle should move smoothly. After injection, gently squeeze the injection site with a cotton ball for 1 minute to prevent bleeding. Then, remove the mouse from its restraints and return it to its original rearing environment.

[0102] The experimental results are as follows:

[0103] The aorta of mice was infected by intravenous injection of DNASE2-overexpressing lentivirus (denoted as DNASE2+ group, indicating injection of lentivirus containing the target gene (viral vector + target gene + marker gene GFP)). The DNASE2- group indicated injection of empty lentivirus without the target gene (containing only the vector backbone + marker gene GFP). This ensured that the infection and integration process of the lentivirus and vector elements (such as promoters and marker genes) would not affect the experimental results. The control group consisted of age-matched mice of the same strain that were not injected with any virus, used to exclude the influence of injection procedures (such as trauma or stress). Figure 26 The results showed that both the DNASE2- and DNASE2+ groups expressed significantly fluorescently labeled proteins (green) in their aortic tissues. Anatomical diagrams of the mouse abdominal aorta were used. Figure 27 ) and ultrasound measurement results of mouse aortic diameter ( Figure 28 The results showed that DNASE2 overexpression significantly reduced the tumorigenesis 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 DNASE2+ group of mice showed a more intact aortic wall structure and fewer elastic fiber breaks, indicating that upregulating DNASE2 expression can slow down the expansion rate of the abdominal aorta in mice and has 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 The results suggest that DNASE2 overexpression effectively cleared cytoplasmic dsDNA in mice, enhancing the resistance of the mouse aorta to angiotensin-induced tumor-like dilation. Simultaneously, the expression level of DNASE2 in the aortic tissue of mice in the DNASE2+ group was increased, while 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 aortic tissue of mice in the DNASE2+ group was significantly higher than that in mice in the DNASE2- group, while the mRNA expression levels of P21, Beclin1, HMGB1, and RIP1 were significantly decreased (P < 0.05). Figure 34 ).

[0104] This invention investigated the mechanism of action of excessively accumulated dsDNA in the cytoplasm of vascular smooth muscle cells (VSMCs) in abdominal aortic aneurysms (AAA) through the above experiments, and the effects of the dsDNA scavenging enzyme DNASE2 on VSMC senescence and AAA. DNASE2 overexpression inhibited the formation of AAA in experimental mice, providing a new target and approach for the preparation of drugs that overexpress the DNASE2 gene or enhance dsDNA scavenging and reduce cytoplasmic dsDNA production, for the prevention or treatment of abdominal aortic aneurysms.

Claims

1. Application of deoxyribonuclease II in the preparation of drugs for the prevention and / or treatment of abdominal aortic aneurysms.

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

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

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

Images