Use of gbp4 gene silencing preparation in preparation of drugs for preventing and treating atherosclerosis

Abstract

The present application relates to the application of GBP4 gene silencing preparation in the preparation of drugs for preventing and treating atherosclerosis, and belongs to the technical field of biological medicine. In order to solve the technical problems that the existing atherosclerosis treatment target is single, cannot target regulate chronic inflammation of blood vessels, and is difficult to suppress disease progression from the root, the present application provides the application of GBP4 gene silencing preparation in the preparation of drugs for preventing and treating atherosclerosis, and the gene silencing preparation is a recombinant lentivirus vector carrying shRNA targeting GBP4. The present application first finds that GBP4 is a positive regulator of atherosclerosis, and the GBP4 gene silencing preparation can reduce the area of atherosclerotic plaque, delay the occurrence and development of atherosclerosis; increase the thickness of the fibrous cap of the plaque, enhance the stability of the plaque, and reduce the risk of plaque rupture; at the same time, reduce the systemic inflammation level, reduce the inflammatory response of the blood vessel wall, and have important clinical application value and broad market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of GBP4 gene silencing agents in the preparation of drugs for the prevention and treatment of atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is a chronic, progressive vasculitis and a major pathological basis for coronary heart disease, cerebral infarction, and peripheral vascular disease. Its pathological features include lipid metabolism disorders, endothelial dysfunction, and chronic inflammatory infiltration of the vascular wall, ultimately leading to arterial wall thickening, lipid plaque formation, and even vascular occlusion. Current mainstream clinical treatments for AS include intensive lipid-lowering with statins, antiplatelet aggregation therapy, and interventional surgery. However, while statins can lower LDL cholesterol levels and stabilize plaques, clinical findings show that some patients still have a high risk of adverse cardiovascular events even after achieving target lipid levels. Furthermore, long-term use of high-dose statins can easily induce toxic side effects such as liver damage, significantly limiting the treatment's effectiveness. Interventional surgery, while rapidly restoring blood supply, cannot reverse the systemic pathological process of AS. Therefore, discovering novel molecular targets independent of lipid metabolism that can directly regulate the inflammatory response of the vascular wall is of significant clinical importance for developing entirely new strategies for the prevention and treatment of AS.

[0003] Guanylate-binding protein 4 (GBP4) is a member of the interferon-induced GTPase family. Existing research shows that GBP4 can regulate the polarization of macrophages towards the pro-inflammatory M1 phenotype, participate in the body's antibacterial immunity and pyroptosis processes, and serve as a biomarker for tumor immunity, playing an important regulatory role in various inflammatory and immune-related diseases.

[0004] Although GBP4 is involved in inflammation regulation, current research focuses on acute lung injury, infection and immunity, and tumors, which are fundamentally different from the chronic vascular inflammatory pathological features of atherosclerosis. Atherosclerosis is a chronic vascular inflammatory disease driven by persistent lipid load, abnormal blood flow shear stress, and metabolic disorders, with a course lasting from months to years. The behavior of immune cells in the plaque microenvironment often differs from that in the acute infection or tumor microenvironment. The same immune regulatory molecule often exhibits functional heterogeneity in different disease microenvironments, and may even play completely opposite regulatory roles. Currently, no research has elucidated the specific role and molecular mechanism of GBP4 in chronic vascular inflammation in atherosclerosis. Therefore, researchers have never considered GBP4 as a potential intervention target for atherosclerosis, representing a significant technological gap. Summary of the Invention

[0005] To address the technical problems of existing atherosclerosis treatments, such as limited target specificity, inability to target and regulate chronic vascular inflammation, and difficulty in fundamentally inhibiting disease progression, this invention provides the application of GBP4 gene silencing agents in the preparation of drugs for the prevention and treatment of atherosclerosis.

[0006] The technical solution of this invention:

[0007] The application of GBP4 gene silencing agents in the preparation of drugs for the prevention and treatment of atherosclerosis, wherein the gene silencing agent is a recombinant lentiviral vector carrying shRNA targeting GBP4, and the nucleotide sequence of the shRNA targeting GBP4 is shown in SEQ ID No. 1.

[0008] Furthermore, the atherosclerosis mentioned is aortic atherosclerosis.

[0009] Furthermore, the aortic atherosclerosis mentioned is aortic root atherosclerosis.

[0010] Furthermore, the aortic root atherosclerosis includes the atherosclerotic plaque stage and / or the fibroatherosclerotic plaque stage.

[0011] Furthermore, the gene silencing agent is in the form of an intravenous injection.

[0012] Furthermore, the viral titer of the recombinant lentiviral vector is 1×10⁻⁶. 8 ~1×10 9 TU / ml.

[0013] The beneficial effects of this invention are:

[0014] This invention is the first to discover that GBP4 is a positive regulator of atherosclerosis, and this is demonstrated through ApoE. - / - A mouse model of atherosclerosis was used to demonstrate for the first time that GBP4 expression level is positively correlated with the severity of atherosclerotic lesions: silencing GBP4 can alleviate the lesions, while overexpressing GBP4 can aggravate the lesions, thus establishing the scientific basis for GBP4 as a new target for the prevention and treatment of atherosclerosis.

[0015] This invention involves tail vein injection of a recombinant lentiviral vector carrying shRNA targeting GBP4, in ApoE. - / -Significant experimental results were achieved in a mouse model. The results showed that, compared with the control group, the LV-sh-GBP4 group mice exhibited significantly reduced aortic plaque area, significantly reduced lipid deposition in the aortic root, significantly increased plaque fibrosis content in the aortic root, and significantly decreased plasma levels of GBP4, inflammatory factors IFN-γ, and IL-6 (P < 0.05). These results indicate that the GBP4 gene silencing agent can reduce atherosclerotic plaque area, delay the development of atherosclerosis, increase plaque fibrosis cap thickness, enhance plaque stability, and reduce the risk of plaque rupture; simultaneously, it reduces systemic inflammation levels and alleviates vascular wall inflammation.

[0016] The GBP4 gene silencing agent proposed in this invention has a mechanism of action independent of existing lipid-lowering drugs, exerting its effect by inhibiting vascular inflammation. The recombinant lentiviral vector enables long-term stable gene silencing, providing sustained efficacy with a single dose and improving patient compliance. In summary, this invention offers a novel treatment strategy for atherosclerosis, possessing significant clinical application value and broad market prospects. Attached Figure Description

[0017] Figure 1 The images show a comparison of the Oil Red O staining results of the aorta in each group of mice in Example 1. A is a photograph of the entire aorta stained with Oil Red O, and B is a comparison of the plaque area ratio.

[0018] Figure 2 The images show a comparison of the pathological staining results of the aortic root of mice in each group in Example 1. A is a photograph of the aortic root stained with HE, Masson's Red and Oil Red O, B is the percentage of plaque area to lumen area, C is the percentage of collagen area to lesion area, and D is the percentage of lipid area to lesion area.

[0019] Figure 3 The image shows a comparison of the plasma GBP4 and inflammatory factor levels in mice in each group in Example 2. A represents the plasma GBP4 level, B represents the plasma IFN-γ level, and C represents the plasma IL-6 level.

[0020] Figure 4 This is a comparison chart of plasma lipid levels in mice of different groups in Example 3. A represents TC, B represents TG, and C represents LDL-C.

[0021] Figure 5 This is a comparison chart of the weight changes of mice in each group in Example 3;

[0022] Figure 6 This is a comparison chart of random blood glucose concentrations in each group of mice in Example 3;

[0023] Figure 7The above is a comparison chart of the oral glucose tolerance test results of mice in each group in Example 3. A is the blood glucose change curve of the oral glucose tolerance test, and B is a comparison chart of quantitative analysis of the area under the curve (AUC).

[0024] Figure 8 Color Doppler echocardiography of mice in each group in Example 4;

[0025] Figure 9 The image shows a comparison of echocardiographic results of mice in different groups in Example 4. A represents left ventricular ejection fraction (EF), B represents left ventricular fractional shortening (FS), C represents left ventricular end-systolic diameter (LVIDs), D represents left ventricular end-diastolic diameter (LVIDd), E represents left ventricular end-diastolic volume (LVEDV), F represents interventricular septal thickness at end-systolic (IVSd), G represents left ventricular posterior wall thickness at end-systolic (LVPWd), and H represents stroke volume (SV).

[0026] Figure 10 The above are comparative images of the Oil Red O staining results of the aorta of mice in each group of Comparative Example 1. A is a photograph of the entire aorta stained with Oil Red O, and B is a comparison of the plaque area ratio.

[0027] Figure 11 The images show a comparison of the pathological staining results of the aortic root of mice in each group in Comparative Example 1. A is a photograph of the aortic root stained with HE, Masson's Red and Oil Red O, B is the percentage of plaque area to lumen area, C is the percentage of collagen area to lesion area, and D is the percentage of lipid area to lesion area.

[0028] Figure 12 The image shows a comparison of the plasma GBP4 and inflammatory factor levels in mice of different groups in Comparative Example 1. A represents plasma GBP4 level, B represents plasma TNF-α level, C represents plasma IL-6 level, and D represents IL-1β level.

[0029] Figure 13 The graph shows a comparison of plasma lipid levels in mice of different groups in Comparative Example 1. A represents total cholesterol (TC), B represents triglycerides (TG), and C represents low-density lipoprotein (LDL-C).

[0030] Figure 14 This is a comparison chart of the weight changes of mice in each group in Comparative Example 1;

[0031] Figure 15 This is a comparison of random blood glucose concentrations in mice of different groups in Comparative Example 1.

[0032] Figure 16 The above is a comparison chart of the oral glucose tolerance test results of mice in each group in Comparative Example 1. A is the blood glucose change curve of the oral glucose tolerance test, and B is a comparison chart of quantitative analysis of the area under the curve (AUC).

[0033] Figure 17Color Doppler echocardiography of mice in each group of Comparative Example 1;

[0034] Figure 18 The following is a comparison of the echocardiographic results of mice in each group in Comparative Example 1: A is EF, B is FS, C is LVIDs, D is LVIDd, E is LVEDV, F is IVSd, G is LVPWd, and H is SV.

[0035] Figure 19 The image shows a comparison of the expression changes of M1 polarization markers after macrophages overexpressed GBP4 in each group in Example 5. A is a Western blot image of GBP4 and INOS proteins, B is a comparison of the expression levels of INOS and GBP4 proteins, and C is a comparison of the mRNA expression levels of M1 macrophage markers (GBP4, INOS, TNF-α, CD86).

[0036] Figure 20 The image shows a comparison of the immunofluorescence staining results of INOS in macrophages after overexpression of GBP4 in each group in Example 5. A is the immunofluorescence staining image of INOS in macrophages, and B is the comparison of the relative fluorescence intensity of INOS in the lesion area.

[0037] Figure 21 This is a comparison of the expression changes of pyroptosis-related proteins in mouse aortic endothelial cells (MAECs) overexpressing GBP4 in each group in Example 5. A is a Western blot image of pyroptosis-related proteins in MAECs, and B is a comparison of the expression levels of pyroptosis-related proteins (NLRP3, GSDMD, ASC, Caspase-1). Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0039] Example 1

[0040] To investigate the intervention effect of tail vein injection of GBP4 silent lentivirus (LV-sh-GBP4) on the degree of atherosclerotic lesions, this embodiment uses ApoE mice as the research subjects and conducts in vivo animal experiments by tail vein injection of LV-sh-GBP4.

[0041] I. Establishment of an animal model of atherosclerosis

[0042] Select male ApoEs aged 6-8 weeks and weighing 18-22g. - / - Mice. A stable mouse model of atherosclerosis was established by feeding mice with a high-fat diet (21% fat, 0.25% cholesterol) for 16 consecutive weeks.

[0043] The male ApoE used in this embodiment - / - Mice were purchased from Liaoning Changsheng Technology Co., Ltd., and high-fat feed was purchased from Jiangsu Medison Biomedical Co., Ltd.

[0044] II. Grouping and Administration Methods

[0045] (1) Grouping method:

[0046] ApoE - / - Mice were randomly divided into four groups:

[0047] NC group: A high-fat diet was followed by the first tail vein injection at the start of the high-fat diet, with a dose of 1×10⁻⁶. 8 Negative control lentivirus at TU / ml was injected once every half month for a total of three injections.

[0048] GBP4 group: A high-fat diet throughout the entire diet, with the first tail vein injection administered at the start of the high-fat diet at a dose of 1×10⁻⁶. 8 TU / ml of silent GBP4 lentivirus, injected once every half month, for a total of three injections;

[0049] Pit group: High-fat diet throughout the entire course, and pitavastatin (Pit) was administered by gavage at the start of the high-fat diet at a dose of 1 mg / kg / day, and was administered throughout the course of the course;

[0050] CD group: The normal diet group served as a control for healthy mice.

[0051] The negative control lentivirus LV-NC and the GBP4 silencing lentivirus LV-sh-GBP4 used in this embodiment were purchased from Shanghai Jikai Gene Co., Ltd. LV-NC carries a disordered interfering sequence and has no targeted silencing effect, used to exclude interference from the lentiviral vector itself on the experimental results. LV-sh-GBP4 carries a specific shRNA sequence targeting the mouse GBP4 gene, which can specifically silence the expression of the GBP4 gene in mice. The nucleotide sequence of the shRNA targeting the mouse GBP4 gene is shown in SEQ ID No. 1, specifically 5'-gaTTCCCTTGTGGAAAGTATT-3'.

[0052] Although ApoE is used in the embodiments of the present invention - / -Mouse was used as the animal model, and the mouse GBP4 gene was used as the target for validation. However, this technique has cross-species applicability and is also applicable to the preparation of human GBP4 genes and human drugs. The reasons are as follows: First, the GBP4 gene is highly conserved in mammals; the amino acid sequence homology between human GBP4 and mouse Gbp4 exceeds 70%, and its functional domains are highly conserved evolutionarily. The shRNA design strategy targeting mouse Gbp4 can be applied equivalently to homologous sequences of the human GBP4 gene. Second, lentiviral vectors and RNA interference mechanisms are highly conserved in mammalian cells. The VSV-G pseudotyped lentivirus used in this embodiment can broadly infect mammalian cells, including humans, and the principle of shRNA silencing target genes is also fully present in human cells. Third, ApoE... - / - Mouse models are internationally recognized as the gold standard for atherosclerosis research. Their pathological characteristics, such as lipid deposition, macrophage infiltration, inflammatory cytokine release, and fibrous cap formation, are highly consistent with those in humans. Therefore, the therapeutic effects validated in this model can be reasonably expected to be equally effective in human patients. In summary, this invention addresses ApoE... - / - The GBP4 gene silencing agents validated in mouse models and their application in the prevention and treatment of atherosclerosis are equivalent to those for human use.

[0053] (2) Method of injecting lentivirus via tail vein:

[0054] Dilute the virus to 1×10 using physiological saline or PBS. 8 TU / ml, use an insulin needle to draw 100~200μl of virus solution, straighten the mouse tail to fully expose the tail vein, and inject smoothly at the tip 1 / 3 of the tail. After injection, remove the needle and apply pressure with a sterile cotton ball to stop bleeding.

[0055] III. Oil Red O staining and detection results of the aorta

[0056] After the experiment, intact aortic tissues from four groups of mice were dissected and fixed in 4% paraformaldehyde solution for 48 hours. Following fixation, the tissues were dehydrated sequentially using 10%, 20%, and 30% sucrose solutions at room temperature for 8 hours, 8 hours, and 12 hours, respectively. The dehydrated aortic tissues were longitudinally dissected ventrally, fixed face up on black gelatin dishes, incubated with 60% isopropanol for 1 minute, and then evenly coated with Oil Red O staining solution. The dishes were then stained in a 37°C water bath in the dark for 20 minutes. After staining, the tissues were rinsed with isopropanol for 1 minute to remove excess stain. The treated aortic tissues were then flattened and fixed, with lipid deposition areas stained red by Oil Red O. Images of the tissue samples were acquired using a high-resolution camera. ImageJ software was used to statistically analyze the Oil Red O-positive staining area and the total aortic area. Graphpad Prism 8 software was used for data analysis and plotting.

[0057] The results are as follows Figure 1 As shown, Oil Red O staining results of the aorta revealed large, continuous red staining areas in the aortic intima of mice in the NC group, indicating significant thickening of the vessel wall. After LV-sh-GBP4 intervention, the red lipid deposition area in the aorta of mice in the GBP4 group was significantly reduced, with positive staining appearing as scattered dots, indicating a substantial decrease in lipid accumulation. The Pit group also showed a significant reduction in aortic lipid deposition. Quantitative statistical results indicated that the plaque area in the aorta of mice in the NC group was approximately 35%, in the GBP4 group it decreased to approximately 15%, and in the Pit group it was approximately 12%. Statistical analysis showed that compared with the NC group, the aortic plaque area in both the GBP4 and Pit groups was significantly reduced (P < 0.001).

[0058] IV. Histopathological staining and detection results of the aortic root tissue

[0059] Four groups of experimental mice were anesthetized and euthanized. Residual blood in the aortic root tissue was rinsed with physiological saline, and intact aortic root samples were obtained. The samples were fixed in 4% paraformaldehyde solution for 48 hours, and then dehydrated sequentially at room temperature using 10%, 20%, and 30% sucrose solutions for 8 hours, 8 hours, and 12 hours, respectively. The dehydrated tissue samples were placed in the center of an embedding cassette, embedding medium was slowly injected, and the cassette was placed at -80℃ for 30 minutes to complete rapid freezing and embedding. Frozen sections were prepared with a section thickness of 6 μm.

[0060] HE staining: Paraffin sections were dewaxed and then sequentially immersed in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, and 75% ethanol for 5 min each. After dewaxing, staining was performed: hematoxylin staining for 2 min, followed by rinsing with distilled water for 15 min; eosin staining for 2 min, followed by rinsing with distilled water for 2 min. After staining, the sections were dehydrated and cleared in a gradient manner, then rinsed with 95% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol for 5 s each, immersed in xylene I for 3 min, and then immersed in xylene II for 5 min. Finally, neutral resin was added for mounting, and the sections were dried overnight in a 60℃ oven. The sections were then observed and photographed under a microscope, and the morphology and area of ​​the plaques were analyzed using ImageJ software.

[0061] Masson staining: The dewaxing process for paraffin sections was the same as for HE staining. After dewaxing, Masson staining was performed: Weiger iron hematoxylin staining for 10 min, followed by rinsing with distilled water for 30 s; differentiation with acidic ethanol solution for 15 s, followed by rinsing with distilled water for 30 s; Masson blue staining with Masson blue solution for 5 min, followed by rinsing with distilled water for 2 min; staining with Ponceau S and acid fuchsin solution for 8 min, followed by rinsing with distilled water for 1 min; rinsing with weak acid working solution for 1 min, immersion in phosphomolybdic acid solution for 2 min, followed by rinsing with weak acid working solution for 1 min again; staining with aniline blue solution for 2 min, followed by rinsing with weak acid working solution for 1 min. After staining, the sections were processed according to the clearing, mounting, and drying procedures for HE staining. Images were observed under a microscope, and the distribution and content of collagen fibers within the plaques were analyzed using ImageJ software. All data were analyzed and plotted using Graphpad Prism 8 software.

[0062] Oil Red O staining: Frozen sections were removed and washed twice with PBS for 10 min each time. After marking the tissue areas with a histochemical pen, the sections were rinsed with washing buffer for 60 s. Sufficient Oil Red O staining solution was added to the tissue surface, completely covering it. The sections were then placed in a 37°C oven in the dark for 40 min, with the staining solution replenished every 10 min to ensure thorough staining. After staining, the sections were thoroughly rinsed with washing buffer and deionized water, blotted dry, and mounted with glycerol gelatin. Tissue images were observed and acquired under a microscope, and the area of ​​Oil Red O positive staining was counted using ImageJ software.

[0063] The results are as follows Figure 2 As shown, the NC group mice exhibited abundant red lipid deposition in the aortic root wall, with large atherosclerotic plaques enriched with foam cells, resulting in severe luminal stenosis. Furthermore, the collagen fibers within the plaques showed light staining, sparse distribution, and thin fibrous caps, indicating poor structural stability. In the GBP4 group, after LV-sh-GBP4 intervention, the area of ​​red lipid deposition in the aortic root was significantly reduced, plaque accumulation was significantly improved, plaque volume was significantly reduced, foam cell infiltration decreased, and luminal stenosis was effectively alleviated. Simultaneously, the blue collagen fiber staining depth within the plaques significantly increased, and the fiber components were more densely and uniformly distributed, effectively thickening the plaque fibrous cap and improving plaque structural stability. The lesion improvement effect in the Pit group mice was consistent with that in the GBP4 group, also exhibiting reduced lipid deposition, smaller plaque volume, and increased collagen fiber content.

[0064] The experimental results of this embodiment demonstrate that silencing GBP4 can significantly reduce the effects induced by a high-fat diet. It reduced the atherosclerotic plaque load in the mouse aorta and aortic root, decreased lipid deposition, alleviated the degree of vascular stenosis, and enhanced plaque stability by increasing the content of collagen fibers in the plaque and thickening the fibrous cap. Its intervention effect was comparable to that of the positive control drug pitavastatin.

[0065] Example 2

[0066] To investigate the intervention effect of GBP4 silencing on systemic inflammatory response in mice with high-fat diet-induced atherosclerosis, this example tested the plasma inflammatory factor levels of mice in each group in Example 1 to evaluate the effect of GBP4 silencing on circulating inflammation levels.

[0067] I. Experimental Methods

[0068] (1) Mouse blood collection:

[0069] Prepare 2ml EDTA anticoagulant tubes in advance. After anesthetizing the mice, collect blood using the ocular blood sampling method. Drop the blood directly into the 2ml EDTA anticoagulant tube, gently invert and mix several times to ensure sufficient contact between the blood and the anticoagulant. Then, place the sample in a centrifuge and centrifuge at 3500 rpm for 15 minutes. After centrifugation, carefully aspirate the supernatant plasma and transfer it to a 1.5ml enzyme-free EP tube. Store at -80℃ for subsequent testing.

[0070] (2) Detection of mouse plasma GBP4 levels by ELISA:

[0071] The concentration of GBP4 in plasma was determined using an ELISA kit provided by Shanghai Jianglai Biotechnology Co., Ltd. Before the experiment, the kit was removed from the refrigerator and allowed to equilibrate at room temperature for 30 minutes. The required number of ELISA strips were prepared, and standard wells, sample wells, and blank wells were set up. 100 μl of different concentrations of GBP4 standard were added to each standard well, 100 μl of the plasma sample to be tested was added to each sample well, and 100 μl of universal diluent was added to each blank well. The strips were sealed with sealing film and incubated at 37°C for 1 hour. After incubation, the liquid in the wells was discarded, and 100 μl of biotin-labeled antibody working solution was added to each well. The plates were resealed and incubated at 37°C for 1 hour. The liquid was discarded, and 250 μl of washing buffer was added to each well. After standing for 30 seconds, the liquid was shaken off, and the plates were patted dry on absorbent paper. This washing process was repeated 3 times. Subsequently, 100 μl of enzyme conjugate working solution was added to each well, and the plates were sealed and incubated at 37°C for 30 minutes. The liquid was discarded, and the plates were washed 5 times. Add 90 μl of TMB substrate solution to each well and incubate at 37°C for 15 min in the dark. Finally, add 50 μl of stop solution to terminate the reaction. Measure the absorbance of each well at 450 nm. Plot a standard curve based on the concentration of the standard and the corresponding absorbance value, and calculate the concentration of GBP4 in each sample using the curve equation.

[0072] (3) ELISA method for detecting plasma IFN-γ and IL-6 levels in mice:

[0073] The levels of IFN-γ and IL-6 in plasma were determined using the appropriate mouse ELISA kit. Before the experiment, the kit was left at room temperature for 30 min, and the required strips were removed. Standard wells, sample wells, and blank wells were prepared. 50 μl of different concentrations of standard were added to each standard well. 10 μl of the plasma sample to be tested was added to each sample well, followed by 40 μl of sample diluent. No reagents were added to the blank wells. Then, 100 μl of detection antibody was added to each of the standard and sample wells. The wells were sealed with sealing film and incubated at 37°C for 1 h. After incubation, the liquid in the wells was discarded, and the plates were patted dry on absorbent paper. 250 μl of washing buffer was added to each well, and after standing for 30 s, the liquid was discarded. This washing process was repeated 5 times. 50 μl of substrate A solution and 50 μl of substrate B solution were added to each well sequentially. The plates were incubated at 37°C for 15 min in the dark. 50 μl of stop solution was added to terminate the reaction, and the absorbance of each well was measured at 450 nm. The concentrations of IFN-γ and IL-6 in each sample were calculated based on the standard curve.

[0074] The results are as follows Figure 3 As shown, compared with the CD group, the plasma GBP4 level in the NC group mice was significantly increased; compared with the NC group, the plasma GBP4 level in the Pit group and the GBP4 group mice was significantly decreased, and the difference was statistically significant, indicating that both pitavastatin and GBP4 silencing intervention can effectively downregulate the expression level of circulating GBP4.

[0075] The plasma IFN-γ level in the NC group mice was significantly higher than that in the CD group; compared with the NC group, the IFN-γ levels in the Pit group and the GBP4 group were significantly decreased, suggesting that both intervention methods can inhibit the secretion of pro-inflammatory factors in atherosclerotic mice.

[0076] The plasma pro-inflammatory factor IL-6 level in the NC group mice was significantly higher than that in the CD group; compared with the NC group, the IL-6 levels in the Pit group and the GBP4 group were significantly lower, indicating that both intervention methods can effectively reduce systemic inflammatory response.

[0077] The experimental results of this embodiment demonstrate that silencing GBP4 can significantly reduce the levels of GBP4 and pro-inflammatory factors IFN-γ and IL-6 in the plasma of atherosclerotic mice, effectively inhibiting the systemic inflammatory response induced by a high-fat diet.

[0078] Example 3

[0079] To investigate the effects of silencing GBP4 on blood lipid levels, body weight changes, and glucose metabolism indicators in mice with high-fat diet-induced atherosclerosis, and to clarify whether its anti-atherosclerotic effect depends on metabolic pathway regulation, this example tested the blood lipid, body weight, and blood glucose-related indicators of mice in each group in Example 1.

[0080] I. Determination of plasma lipid levels in mice using the microplate method

[0081] Using the test kits provided by Edison Biotechnology Co., Ltd., plasma total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels were measured by the microplate method.

[0082] Total cholesterol content determination: In a 96-well plate, add 2.5 μl of the plasma sample to be tested and 2.5 μl of TC standard, add 2.5 μl of distilled water to the blank well, and then add 250 μl of reagent I to each well. After mixing thoroughly, incubate at 37℃ for 10 min, and measure the absorbance value of each well at a wavelength of 510 nm.

[0083] Triglyceride content determination: Add 20 μl of standard and 20 μl of test sample to each well of a 96-well plate, and do not add any to the blank wells; then add 20 μl of reagent I and 80 μl of reagent II to each well in sequence, and add only 100 μl of reagent II to the blank wells; then add 20 μl of reagent III and 60 μl of reagent IV, and incubate at room temperature in the dark for 30 min. Measure the absorbance of each well at a wavelength of 510 nm.

[0084] Determination of low-density lipoprotein cholesterol (LDL-C) content: Add 2.5 μl of the test sample and 2.5 μl of LDL-C standard to each well of a 96-well plate, and add 2.5 μl of distilled water to the blank well. Add 180 μl of reagent one to each well, mix thoroughly, and incubate at 37°C for 5 min. Read the absorbance value A1 at 546 nm. Then add 60 μl of reagent two, mix thoroughly, and incubate at 37°C for 10 min. Read the absorbance value A2 at 546 nm again. Calculate the LDL-C concentration of the sample based on the difference between the two absorbance values.

[0085] The results are as follows Figure 4 As shown, plasma TC, TG, and LDL-C levels were significantly elevated in the NC group mice; compared with the NC group, there were no significant differences in TC, TG, and LDL-C levels in the GBP4 group and the Pit group mice (P > 0.05). This indicates that silencing GBP4 does not affect ApoE. - / - The blood lipid levels in mice, i.e., the anti-atherosclerotic effect of the GBP4 silencing agent, were independent of the lipid-lowering pathway.

[0086] II. Mouse weight detection

[0087] From the start of modeling, the weight of each group of mice was measured every two weeks using an electronic scale, and the data was recorded and a weight change curve was plotted.

[0088] The results are as follows Figure 5 As shown, during the 16-week experimental period, the body weight of mice in each group showed a steady upward trend, with no significant differences between groups.

[0089] III. Random blood glucose detection in mice

[0090] Mice underwent random blood glucose testing every four weeks. Before testing, mice were fasted for 8 hours but allowed free access to water. After fasting, fasting blood glucose levels were measured and recorded using a blood glucose meter via tail vein sampling.

[0091] The results are as follows Figure 6 As shown, the blood glucose levels of mice in each group were within the normal range, and there was no statistically significant difference between the groups.

[0092] IV. Oral glucose tolerance test

[0093] Mice were fasted for 8 hours and then given free access to water. Blood was collected from the tail vein to measure fasting blood glucose levels. Subsequently, 50% glucose solution was administered by gavage at a dose of 2 g / kg. Blood glucose levels were measured by tail vein at 15, 30, 60, 90, and 120 minutes after gavage, and blood glucose curves were plotted.

[0094] The results are as follows Figure 7 As shown, the blood glucose change trends of mice after gavage administration of glucose were consistent across groups, with no significant difference in blood glucose levels between groups.

[0095] The experimental results of this embodiment demonstrate that silencing GBP4 has no significant effect on blood lipids, body weight, and glucose metabolism indicators in mice with high-fat diet-induced atherosclerosis, and its anti-atherosclerotic effect is independent of lipid-lowering and glucose metabolism regulation pathways.

[0096] Example 4

[0097] To investigate the effect of silencing GBP4 on cardiac function in mice with high-fat diet-induced atherosclerosis, color Doppler echocardiography was performed on mice in each group in Example 1 to assess cardiac contraction and structural related indicators.

[0098] Before euthanizing the mice in week 16 of the experiment, all mice in each group were anesthetized, and their chest hair was removed using depilatory cream to fully expose the detection area. The mice were placed on the operating table, fixed in a left lateral decubitus position, and an appropriate amount of ultrasound coupling agent was evenly applied to the prepared area. The ultrasound probe was placed on the mouse's chest, and color Doppler echocardiography was used to record key cardiac function indicators such as left ventricular ejection fraction (LVEF), fractional shortening (FS), and left ventricular diameter. Subsequent data statistical analysis was performed using software.

[0099] The results are as follows Figure 8 and Figure 9 As shown, the left ventricular ejection fraction and left ventricular diameter of mice in the NC group were within the normal range; compared with the NC group, there were no significant differences in left ventricular ejection fraction, fractional shortening, and left ventricular diameter between the GBP4 group and the Pit group (P>0.05). These results indicate that silencing GBP4 has no significant effect on cardiac function in mice with high-fat diet-induced atherosclerosis.

[0100] The experimental results of this embodiment demonstrate that silencing GBP4, while exerting its anti-atherosclerotic effect, does not adversely affect the cardiac contractile function or ventricular structure of atherosclerotic mice, suggesting that this intervention method has good cardiac safety.

[0101] Comparative Example 1

[0102] To investigate the effect of macrophage-specific overexpression of GBP4 on high-fat diet-induced [effects / conditions]. To investigate the effects of GBP4 overexpression on atherosclerotic lesions in mice, a macrophage-specific GBP4 overexpression model was constructed in a comparative manner to evaluate its effects on atherosclerotic lesions in the aorta and aortic root.

[0103] I. Grouping and Administration Methods

[0104] ApoE - / - Mice were randomly divided into three groups:

[0105] AAV6-NC group: A high-fat diet throughout, with a dose of 1×10⁻⁶ administered via tail vein injection at the start of the high-fat diet. 11 TU / ml control virus AAV6-NC;

[0106] AAV6-OE-GBP4 group: A high-fat diet throughout the entire course, with an equivalent dose of 1×10⁻⁶ administered via tail vein injection at the start of the high-fat diet. 11 AAV6-OE-GBP4 adeno-associated virus at TU / ml;

[0107] CD group: The normal diet group served as a control for healthy mice.

[0108] The adeno-associated virus (AAV6-OE-GBP4) carrying the F4 / 80 macrophage-specific promoter and the control virus AAV6-NC used in this comparative example were both purchased from Shanghai Jikai Gene Co., Ltd. The nucleotide sequence of the F4 / 80 macrophage-specific promoter is shown in SEQ ID No. 2. After AAV6-OE-GBP4 was injected into mice via the tail vein, it initiated GBP4 gene expression only in macrophages, allowing macrophages to overexpress the GBP4 protein. The control virus AAV6-NC was an empty vector control virus without the GBP4 gene expression element, used to exclude the influence of the virus itself on the experimental results.

[0109] II. Comparison of the degree of atherosclerosis in mice of different groups after macrophage-specific overexpression of GBP4

[0110] After feeding mice with a high-fat diet for 16 weeks, aortic tissue was collected from each group for Oil Red O staining, Oil Red O staining of the aortic root, HE staining, and Masson staining. The specific experimental methods were the same as in Example 1.

[0111] Oil Red O staining results of the aorta are as follows Figure 10 As shown, scattered red-stained areas were visible in the aortic intima of mice in the AAV6-NC group, with smaller plaque areas. Compared with the AAV6-NC group, the red-stained areas in the aorta of mice in the AAV6-OE-GBP4 group were significantly increased, and the plaque area was significantly enlarged. These results indicate that macrophage-specific overexpression of GBP4 can significantly increase ApoE... - / - Aortic plaque area in mice.

[0112] Oil Red O staining, HE staining, and Masson staining of the aortic root are as follows: Figure 11 As shown, in the AAV6-NC group mice, a small amount of red lipid deposition was visible in the vessel wall of the aortic root, the plaque area was small, and small atherosclerotic plaques were visible. The degree of luminal stenosis was mild, and more blue collagen fiber staining was visible, indicating that the fibrous cap was thicker and the plaque stability was better. Compared with the AAV6-NC group, the red lipid deposition area in the aortic root of the AAV6-OE-GBP4 group mice was significantly increased, the plaque area was enlarged, the degree of luminal stenosis was more severe, and the blue collagen fiber staining in the plaque was significantly reduced and sparsely distributed, indicating that the fibrous cap was thinner and the plaque stability was decreased.

[0113] The above staining results indicate that macrophage-specific overexpression of GBP4 can significantly increase ApoE. - / - Lipid deposition and plaque area in the aortic root of mice were significantly reduced, plaque fibrosis content was decreased, and plaque stability was reduced.

[0114] III. Comparison of plasma inflammatory factors in mice after macrophage-specific overexpression of GBP4

[0115] The levels of GBP4, TNF-α, IL-6, and IL-1β in the plasma samples of mice in each group were determined using ELISA. The specific experimental method was the same as in Example 2.

[0116] The results are as follows Figure 12 As shown, compared with the AAV6-NC group mice, the levels of inflammatory factors such as TNF-α and IL-6 in the plasma of the AAV6-OE-GBP4 group mice were significantly increased (P < 0.05). This indicates that macrophage-specific overexpression of GBP4 can significantly increase ApoE levels. - / - Levels of pro-inflammatory factors TNF-α and IL-6 in mouse plasma.

[0117] IV. Comparison of plasma lipids in mice after macrophage-specific overexpression of GBP4

[0118] The plasma levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in mice of each group were determined using the microplate method. The specific experimental method was the same as in Example 3.

[0119] The results are as follows Figure 13 As shown, there were no significant differences in TC, TG, and LDL-C levels between the AAV6-NC group and the AAV6-OE-GBP4 group (P > 0.05). This indicates that macrophage-specific overexpression of GBP4 does not affect ApoE. - / - In mice, the blood lipid levels, specifically the role of GBP4 in promoting atherosclerosis, were independent of the lipid-lowering pathway.

[0120] V. Comparison of body weight, random blood glucose, and glucose tolerance in mice of different groups after macrophage-specific overexpression of GBP4

[0121] Mice were weighed every two weeks using an electronic scale. Random blood glucose levels were measured every four weeks, and an oral glucose tolerance test was performed at the end of the experiment. The specific experimental methods were the same as in Example 3.

[0122] The results are as follows Figure 14 , Figure 15 and Figure 16 As shown, the body weight of mice in both the AAV6-NC and AAV6-OE-GBP4 groups showed a steady upward trend during the 16-week experimental period, with no significant differences between the groups. Random blood glucose testing results showed that blood glucose levels in all groups were within the normal range, with no significant differences between groups. Oral glucose tolerance tests showed that blood glucose levels in both groups reached their peak 30 minutes after glucose administration, and then gradually decreased to baseline levels, with no significant differences between groups. This indicates that macrophage-specific overexpression of GBP4 does not affect ApoE. - / - Mouse body weight, random blood glucose, and glucose tolerance.

[0123] VI. Comparison of cardiac function among mice in different groups after macrophage-specific overexpression of GBP4

[0124] Mice in each group were anesthetized, their chest hair was removed, and they were placed on the operating table in a left lateral decubitus position. An ultrasound probe was placed in the mouse's chest to perform cardiac ultrasound examination, and parameters such as left ventricular ejection fraction and left ventricular diameter were recorded. The specific experimental method was the same as in Example 4.

[0125] The results are as follows Figure 17 and Figure 18 As shown, compared with the AAV6-NC group, there were no significant differences in left ventricular ejection fraction and left ventricular diameter in the AAV6-OE-GBP4 group mice (P>0.05). This indicates that macrophage-specific overexpression of GBP4 does not affect ApoE. - / - Heart function in mice.

[0126] Example 5

[0127] To verify the cellular mechanism by which GBP4 overexpression promotes atherosclerosis, this embodiment conducted an in vitro experiment on primary bone marrow macrophages (BMDM) overexpressing GBP4 to observe its effect on macrophage M1 polarization, and observed its effect on pyroptosis of vascular endothelial cells through co-culture experiments.

[0128] I. Experimental Materials

[0129] Primary bone marrow macrophages were extracted from the tibia and femur of male C57BL / 6 mice aged 6-8 weeks. The C57BL / 6 mice weighed 18-22g and were purchased from Liaoning Changsheng Technology Co., Ltd.

[0130] Mouse aortic endothelial cell line (MAECs) was purchased from Pronoss, and the Jet-Prime transfection kit was purchased from Shanghai Newqi Health Technology Co., Ltd. GBP4 overexpression plasmid and control plasmid were purchased from Harbin Jingwei Encyclopedia Biotechnology Co., Ltd. The GBP4 overexpression plasmid carries the mouse Gbp4 gene coding sequence (GenBank accession number: NM_001350919.1), while the control plasmid is an empty vector plasmid that does not carry the mouse Gbp4 gene coding sequence. This was used to exclude the non-specific influence of the plasmid backbone and transfection operation itself on the experimental results.

[0131] The complete culture medium for mouse MAECs consisted of DMEM high glucose medium, 10% (v / v) fetal bovine serum, 100 IU / mL penicillin and 100 mg / mL streptomycin.

[0132] The complete culture medium for BMDM (Bone Marrow Derived Macrophages) consists of RIPM-1640 medium, 10% (v / v) fetal bovine serum, 100 IU / ml penicillin, and 100 mg / ml streptomycin.

[0133] The BMDM differentiation culture medium was prepared by adding M-CSF (20 μg / ml) to the complete BMDM culture medium.

[0134] II. Effects of GBP4 overexpression on macrophage M1 polarization

[0135] (I) Extraction and culture of primary bone marrow macrophages

[0136] Eight-week-old male C57BL / 6 mice were anesthetized with afotin (0.25 g / kg). The mice were thoroughly disinfected by immersion in 75% alcohol. The mice were fixed in a supine position, and the skin of the abdomen and both hind limbs was cut open to expose the muscles. The muscles at the hip joint were cut open, as were the hip and ankle joints. The hind limbs were removed and immersed in a dual-antibody solution. The knee ligaments were severed, and the femur and tibia were severed, with muscle tissue removed. After removing the fibula, the femur and tibia were immersed in the dual-antibody solution again for 5 minutes, and any remaining muscle tissue was removed by rubbing with gauze. The femur and tibia were placed in the dual-antibody solution, and the epiphyses at both ends were removed, leaving the diaphysis intact.

[0137] Using a 1ml syringe, draw 1ml of RIPM-1640 culture medium and flush the bone marrow cavity from one end until the color changes from red to white. Repeat the same process from the other end, collecting the cell suspension. Filter the cell suspension through a 70μm filter and centrifuge at 500g / min for 5 min at room temperature. Discard the supernatant, add erythrocyte lysis buffer, let stand for 5 minutes, centrifuge again at 500g / min for 5 min, and discard the supernatant.

[0138] Resuspend the cells in differentiation culture medium, seed them into six-well plates containing coverslips, and incubate them in an incubator. After three days, add 1 ml of fresh differentiation culture medium to the six-well plates. Replace the medium with fresh differentiation culture medium on the fifth day, and replace it with complete culture medium on the sixth day. After incubating overnight, subsequent experiments can be performed.

[0139] (II) Cell transfection

[0140] BMDM at 10 per well 5 Cells were seeded at a density of 70-80% in 6-well plates and transfected when confluence reached 70-80%. The Jet-Prime transfection kit was used to transfect cells with both the control plasmid (NC) and the GBP4 overexpression plasmid (OE-GBP4). Before transfection, the old culture medium was removed and replaced with 1.8 ml of serum-free medium. 200 μl of pre-mixed transfection reagent, transfection solvent, and either the overexpression plasmid or control plasmid solution were added to each well. The 6-well plates were incubated for 4-6 hours. After incubation, the medium was replaced with complete medium, and incubation continued for 18-24 hours. 24 hours after transfection, LPS and IFN-γ were added to induce differentiation of cells into M1 macrophages.

[0141] (III) Western blot detection

[0142] Cells were harvested 48 hours after transfection, and each group of cells was lysed with lysis buffer. After homogenization, the supernatant was collected by centrifugation, and the protein content was measured using a BCA kit. SDS-PAGE electrophoresis was performed with 50 μg of protein. Proteins were transferred to PVDF membranes using the wet transfer method; the membranes were blocked with 50 g / L skim milk powder solution at room temperature for 2 h, and then primary antibodies (GBP4, iNOS, β-actin) were added and the membranes were gently shaken overnight at room temperature; the membranes were washed with TBST, and HRP-labeled secondary antibody was added, followed by gentle shaking at room temperature for 1.5 h; the membranes were thoroughly washed with TBST, and ECL chromogenic buffer was added. The membranes were then photographed using an automated gel analyzer, and the relative expression levels of the corresponding proteins were obtained by dividing the gray value of the corresponding protein band by the gray value of the internal control β-actin.

[0143] (iv) qRT-PCR detection of M1 macrophage marker mRNA expression

[0144] BMDM cells from each group were collected, and total RNA was extracted using Trizol reagent. RNA concentration and purity were measured using a spectrophotometer. RNA samples with an A260 / A280 ratio between 1.8 and 2.0 were used for subsequent experiments. 1 μg of total RNA was used to synthesize cDNA using a reverse transcription kit. The cDNA obtained from reverse transcription was used as a template for qRT-PCR amplification using the SYBR Green method. The reaction mixture consisted of 10 μl: 5 μl SYBR Green Master Mix, 0.5 μl each of forward and reverse primers (10 μM), 2 μl cDNA template, and RNase-free water to a final volume of 10 μl. The amplification program was: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds; 60℃ annealing for 30 seconds, for a total of 40 cycles. β-actin was used as an internal control gene, and 2... -ΔΔCt The relative expression levels of the target gene were calculated. M1 macrophage markers detected included INOS, TNF-α, and CD86.

[0145] The results are as follows Figure 19 As shown, compared with the NC group, the protein and mRNA levels of GBP4 in macrophages of the OE-GBP4 group were significantly increased, proving that the overexpression model was successfully constructed.

[0146] Meanwhile, the protein and mRNA expression levels of M1 polarization markers such as INOS, TNF-α, and CD86 were also significantly upregulated, suggesting that macrophage overexpression of GBP4 can promote macrophages to pro-inflammatory M1 polarization and enhance the inflammatory response.

[0147] (v) Immunofluorescence staining

[0148] Cell slides were removed from a -80°C freezer and washed twice with PBS, 10 min each time. Cells were fixed in 4% paraformaldehyde for 10 min, then the slides were immersed in PBST and washed 6 times with gentle shaking, 5 min each time. Cells were permeabilized with 1% Triton X-100 at room temperature for 10 min, followed by 6 washes with PBST, 5 min each time. The cell surface was covered with goat serum and blocked at room temperature for 1 hour. iNOS primary antibody was added to cover the cells, and the cells were incubated overnight at 4°C. Cells were washed 6 times with gentle shaking, 5 min each time, with PBST. Fluorescent secondary antibody solution was added, and the cells were incubated in a humidified chamber at room temperature in the dark for 1 hour. Cells were washed 6 times with gentle shaking, 5 min each time, with PBST. DAPI staining was added for 5 min. Cells were washed 4 times with gentle shaking, 5 min each time, and mounted with an anti-quenching agent. The slides were photographed under a fluorescence microscope, and the fluorescence intensity was analyzed using ImageJ software.

[0149] The results are as follows Figure 20 As shown, compared with the NC group, the OE-GBP4 group showed a significantly enhanced green fluorescence signal of INOS in macrophages, and a significantly increased number of positively stained cells. Quantitative analysis showed that the relative fluorescence intensity of INOS in the OE-GBP4 group was significantly higher than that in the NC group (P < 0.0001), further confirming that overexpression of GBP4 can significantly upregulate the expression of the M1 marker INOS and promote pro-inflammatory polarization of macrophages.

[0150] III. Effects of GBP4-overexpressing macrophages on pyroptosis of vascular endothelial cells

[0151] (a) Cell grouping and transfection

[0152] After extracting BMDM, first place a coverslip in a six-well plate and inoculate with BMDM. After 6-7 days of culture, when the BMDM confluence reaches 70-80%, divide the BMDM into two groups: the NC group (transfected with the control plasmid) and the OE-GBP4 group (transfected with the GBP4 overexpression plasmid). Transfection is performed using the Jet-Prime transfection kit, following the same steps as above. 24 hours after transfection, change the culture medium and continue culturing for another 24 hours.

[0153] (ii) Co-culture of BMDM and MAECs

[0154] The supernatant of each group of BMDM was collected and added to mouse aortic endothelial cells (MAECs) for 48 hours.

[0155] (III) Western blot detection

[0156] MAECs were collected, and the expression levels of pyroptosis-related proteins (NLRP3, GSDMD, ASC, Caspase-1) were detected by Western blot, following the same steps as above.

[0157] The results are as follows Figure 21 As shown, compared with MAECs co-cultured in the NC group, the expression levels of pyroptosis-related proteins NLRP3, GSDMD, ASC, and Caspase-1 in MAECs co-cultured in the OE-GBP4 group were significantly increased. These data indicate that overexpression of GBP4 in BMDM can promote pyroptosis of vascular endothelial cells.

[0158] The experimental results of this embodiment show that overexpression of GBP4 in mouse BMDM promotes macrophage polarization towards the M1 type and leads to pyroptosis of endothelial cells. This suggests that GBP4 may exacerbate atherosclerotic lesions through a dual mechanism of regulating macrophage polarization and endothelial cell pyroptosis.

Claims

1. The application of GBP4 gene silencing agents in the preparation of drugs for the prevention and treatment of atherosclerosis, characterized in that, The gene silencing agent is a recombinant lentiviral vector carrying shRNA targeting GBP4, and the nucleotide sequence of the shRNA targeting GBP4 is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The aforementioned atherosclerosis is aortic atherosclerosis.

3. The application according to claim 2, characterized in that, The aortic atherosclerosis mentioned refers to atherosclerosis at the aortic root.

4. The application according to claim 3, characterized in that, The aortic root atherosclerosis includes the atherosclerotic plaque stage and / or the fibro-atherosclerotic plaque stage.

5. The application according to claim 4, characterized in that, The gene silencing agent is administered intravenously.

6. The application according to claim 5, characterized in that, The viral titer of the recombinant lentiviral vector is 1×10⁻⁶. 8 ~1×10 9 TU / ml.

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