Application of 2-hydroxyisobutyrylation modification of PRDX2-K10 site in prevention and treatment of restenosis after vascular injury

Abstract

This invention discloses the application of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the prevention and treatment of restenosis after vascular injury. In this invention, the reduction of Khib at the PRDX2-K10 site weakens the inhibitory effect of PRDX2 on oxidative stress and inflammatory response, thereby promoting abnormal proliferation, migration and inflammatory response of VSMCs through the ROS-NF-κB axis. By simulating the maintenance or enhancement of the Khib state of PRDX2-K10, the pathological phenotype of VSMCs can be improved and the formation of restenosis after vascular injury can be reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the prevention of restenosis after vascular injury. Background Technology

[0002] Restenosis refers to the phenomenon where blood vessels or cavities narrow again after surgery or interventional treatment (such as stent placement or balloon dilation) due to abnormal local tissue proliferation or inflammatory response. It is common after cardiovascular interventional procedures, but can also occur in the biliary tract, digestive tract, and other locations, requiring intervention through a combination of medication, secondary surgery, or lifestyle adjustments.

[0003] Following endovascular treatment for lower extremity arteriosclerosis obliterans, in-stent restenosis (ISR) remains one of the major issues affecting long-term patency. Its pathological basis lies in the phenotypic transformation, abnormal proliferation and migration, and neointimal formation of vascular smooth muscle cells (VSMCs) after vascular injury. Increasing research indicates that post-translational modifications play a crucial regulatory role in vascular remodeling. Therefore, studying the changes in Khib during restenosis is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide an application of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the prevention and treatment of restenosis after vascular injury. In this invention, the reduction of Khib at the PRDX2-K10 site weakens the inhibitory effect of PRDX2 on oxidative stress and inflammatory response, thereby promoting abnormal proliferation, migration and inflammatory response of VSMCs through the ROS-NF-κB axis. By simulating the maintenance or enhancement of the Khib state of PRDX2-K10, the pathological phenotype of VSMCs can be improved and the formation of restenosis after vascular injury can be reduced.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides the application of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of products that inhibit VSMC phenotypic transformation.

[0006] A second aspect of the invention provides the use of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of products that inhibit the proliferation and / or migration of VSMCs.

[0007] The third aspect of this invention provides the application of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of drugs for preventing restenosis after vascular injury.

[0008] The fourth aspect of the present invention provides the application of a PRDX2-K10 site 2-hydroxyisobutyrylation modification mimic in the preparation of a product that inhibits the phenotypic transformation of VSMCs, wherein the PRDX2-K10 site 2-hydroxyisobutyrylation modification mimic is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

[0009] The fifth aspect of the present invention provides the use of a PRDX2-K10 site 2-hydroxyisobutyrylation modification mimic in the preparation of a product that inhibits the proliferation and / or migration of VSMCs, wherein the PRDX2-K10 site 2-hydroxyisobutyrylation modification mimic is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

[0010] The sixth aspect of the present invention provides the use of a PRDX2-K10 site 2-hydroxyisobutyrylation mimic in the preparation of a drug for preventing restenosis after vascular injury, wherein the PRDX2-K10 site 2-hydroxyisobutyrylation mimic is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

[0011] The seventh aspect of the present invention provides the application of a recombinant vector expressing a mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of a product that inhibits the phenotypic transformation of VSMCs, wherein the mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation modification is obtained by mutating the 10th K lysine to threonine T in the amino acid sequence of the PRDX2 protein.

[0012] The eighth aspect of the present invention provides the use of a recombinant vector expressing a mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of a product that inhibits the proliferation and / or migration of VSMCs, wherein the mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation modification is obtained by mutating the 10th K lysine to threonine T in the amino acid sequence of the PRDX2 protein.

[0013] The ninth aspect of the present invention provides the use of a recombinant vector expressing a mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of a drug for preventing restenosis after vascular injury, wherein the mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation modification is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

[0014] The tenth aspect of the present invention provides a drug for preventing restenosis after vascular injury, the drug comprising a mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation and / or a recombinant vector expressing the mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation. The PRDX2-K10 site 2-hydroxyisobutyrylation modification mimicry is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

[0015] In this invention, the recombinant vector expressing the PRDX2-K10 site 2-hydroxyisobutyrylation modification mimic is constructed from the nucleic acid encoding the PRDX2-K10 site 2-hydroxyisobutyrylation modification mimic and the expression vector; preferably, the expression vector is adeno-associated virus, specifically adeno-associated virus type 9.

[0016] Preferably, the nucleic acid sequence encoding the PRDX2-K10 site 2-hydroxyisobutyrylated modification mimic is obtained by replacing the codon AAA encoding the 10th lysine of PRDX2 with the codon ACC encoding threonine, based on the PRDX2 protein-encoded nucleic acid sequence.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention demonstrates that, compared to healthy blood vessels, the overall level of Pan Khib in ISR specimens was significantly reduced; a consistent decreasing trend was also observed in rat carotid balloon-injured vessels and PDGF-BB-treated VSMCs, suggesting a relatively stable state of low Khib expression after vascular injury. Khib modification proteomics analysis revealed that the PRDX2-K10 site is a key differential modification site, and this site is highly conserved across different species. Co-IP combined with Western blotting further confirmed that PRDX2-related Khib levels decreased in VSMCs after vascular injury and PDGF-BB stimulation. Functional experiments showed that under PDGF-BB induction, K10T significantly reduced intracellular ROS levels and the expression of IL-6, MCP-1, and ICAM-1, and inhibited VSMC proliferation and migration. WT had a certain protective effect, while the protective effect of K10R was significantly weakened. The changes in the expression of MMP2, MMP9, PCNA, and Cyclin D1 were consistent with the above results. Mechanistic studies showed that exogenous ROS could partially weaken the protective effect of K10T, and NAC could improve the high ROS and high proliferation and migration phenotype in the K10R state. Transcriptome analysis revealed significant differential expression profiles between the K10T and K10R treatment groups. Enrichment analysis indicated significant alterations in pathways affecting VSMC phenotypes, including TNF, IL17, chemokines, and NF-κB. Western blotting further confirmed that the PRDX2-K10 site status influences NF-κB signaling activation by regulating ROS. In vivo experiments showed that adeno-associated virus type 9 (AAV9)-mediated PRDX2-K10T expression significantly reduced neointimal hyperplasia and luminal stenosis after balloon injury, decreased ROS levels in the vessel wall, and reduced the expression of IL-6, MCP-1, and ICAM-1. The WT group also showed some improvement, while the improvement in the K10R group was more limited. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0019] Figure 1 The expression levels of Pan-Khib in human ISR and rat injured blood vessels in the embodiments of the present invention; Figure 2 In this embodiment of the invention, the combined analysis of Khib modifier proteomics and proteomics identified PRDX2 K10 as a key modification site. Figure 3 This illustrates the in vitro and in vivo pathological stimulation-induced downregulation of PRDX2 Khib levels and overall Khib levels in embodiments of the present invention. Figure 4 This invention describes the construction of the PRDX2 K10 site-directed mutant and its effects on the expression of ROS and inflammatory factors in VSMCs. Figure 5 This is the result of a study in the embodiments of the present invention simulating the suppression of the expression of proteins related to VSMC proliferation and migration by maintaining the Khib state at the PRDX2 K10 site; Figure 6 The results of this study, conducted in an embodiment of the invention, demonstrate that maintaining the Khib state at the PRDX2 K10 site significantly inhibits the proliferation and migration of VSMCs. Figure 7 This is the result of a study in this invention showing that exogenous ROS (H2O2) can partially reverse the inhibitory effect of the K10T mutant on the proliferation and migration of VSMCs; Figure 8 This is the result of the study in this invention on how the ROS scavenger (NAC) can partially reverse the abnormal proliferation and migration of VSMCs mediated by the K10R mutant; Figure 9 This is a study result from an embodiment of the present invention on the regulation of inflammation, proliferation and migration of VSMCs by PRDX2 K10 Khib through the ROS / NF-κB axis; Figure 10 This is the result of a study in this invention showing that AAV9-mediated PRDX2-K10T expression significantly improved restenosis in rats after carotid artery injury in vivo. Detailed Implementation

[0020] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0021] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Example This example studies the therapeutic effect of PRDX2-K10 site 2-hydroxyisobutyrylation modification on restenosis after vascular injury: 1. Experimental materials: 1.1 Clinical specimen sourcing and ethical approval A total of 10 vascular specimens from patients with in-stent restenosis (ISR) and 10 vascular specimens from healthy controls were collected. The ISR specimens were all from the diseased vascular tissue of patients who underwent amputation due to severe lower limb ischemia and were confirmed to have ISR by computed tomography angiography (CTA) during the preoperative follow-up. The diagnosis of ISR was based on relevant literature [4] and combined with the patient's clinical manifestations and imaging results. The inclusion criteria were as follows: (1) age ≥18 years; (2) previous femoral artery stent implantation; (3) recurrence or aggravation of lower limb ischemia-related symptoms during the follow-up period; (4) ankle-brachial index (ABI) <0.9 and CTA showed that the degree of in-stent stenosis was >50%; (5) able to cooperate in completing the follow-up and imaging examination after treatment. The exclusion criteria were as follows: (1) those who refused to undergo CTA re-examination; (2) those with more than two ISRs and who received revascularization therapy again within one month after the diagnosis of ISR; (3) those who did not receive antiplatelet therapy regularly; (4) those with severe heart failure with a left ventricular ejection fraction of <40%; (5) those with a history of acute myocardial infarction; (6) those with autoimmune diseases, connective tissue diseases or malignant tumors; and (7) those with dysfunction of important organs. The vascular specimens of healthy controls were obtained from the abdominal aortic tissue of healthy donors that were discarded after being trimmed during organ transplantation surgery in our hospital. After the clinical tissue specimens were collected from the body, they were immediately rinsed 1-2 times with pre-cooled physiological saline to remove surface blood, and then quickly frozen in liquid nitrogen and properly stored in an ultra-low temperature freezer at -80℃. Given the limited quantity of clinical tissue specimens, and the need for some samples to be used for routine pathological preservation, tissue storage, and subsequent backup, 5 specimens were randomly selected from each group of 10 for molecular biological testing such as protein expression analysis and immunofluorescence staining. The procedures for human specimen collection and the use of clinical data have all been rigorously approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University (Ethics Approval No.: QYFYWZLL30916). All enrolled patients were fully informed before surgery that their surgical specimens and clinical pathology data would be used for scientific research, and signed written informed consent forms according to standard procedures. All rights and interests of the participants were fully protected, and the entire research process strictly adhered to the Declaration of Helsinki and other relevant medical ethical guidelines.

[0023] 1.2 Approval of Laboratory Animals and Ethics Healthy male Sprague-Dawley (SD) rats, weighing 350–400 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. All experimental animals were housed in SPF (Specific Pathogen Free) grade standard animal rooms, maintaining a constant temperature (22±2℃) and relative humidity (50%–60%), using a 12-hour light / 12-hour dark circadian rhythm. During this period, animals had free access to standard maintenance feed and sterilized drinking water. The animal experimental protocol and handling procedures for this project have been rigorously approved by the Laboratory Animal Welfare and Ethics Committee (Ethics Approval No.: NO. 20250501SD9920250901035). All animal experimental procedures strictly adhered to laboratory animal welfare and ethical guidelines and relevant management regulations to minimize the suffering of the experimental animals.

[0024] 1.3 Cells The primary human aortic vascular smooth muscle cells (HA-VSMCs, hereinafter referred to as VSMCs) used in the in vitro experiments were kindly donated by Professor Yu Tao of the Institute of Translational Medicine, Affiliated Hospital of Qingdao University. To ensure that the primary cells maintained a good differentiation state and smooth muscle cell-specific phenotype, and to avoid phenotypic transformation and aging caused by excessive passage, all in vitro intervention experiments uniformly used cells from passage 3 to 8.

[0025] 1.4 Main Antibodies and Reagents The main antibodies and reagents used in this invention are shown in Table 1.

[0026] Table 1 Primary Antibody Pan-Khib (Anti-2-Hydroxyisobutyryllysine, Mouse mAb) Jingjie Biotechnology PTM-802 WB 1:1000; IF 1:50 Primary Antibody PRDX2(Peroxiredoxin 2Polyclonal antibody) Wuhan Sanying 10545-2-AP WB 1:4000; Co-IP / WB verification Primary Antibody Flag(DYKDDDDK tagRecombinant monoclonalantibody) Wuhan Sanying 80801-2-RR WB 1:10000 Primary Antibody α-SMA (Alpha smoothmuscle actin specificRecombinant monoclonalantibody) Wuhan Sanying 80008-1-RR WB 1:100000; IF 1:1000 Primary Antibody MMP2 (Polyclonal antibody) Wuhan Sanying 10373-2-AP WB 1:800 Primary Antibody MMP9 (N-terminal Polyclonal antibody) Wuhan Sanying 10375-2-AP WB 1:1500 Primary Antibody PCNA (Polyclonal Antibody) Wuhan Sanying 10205-2-AP WB 1:10000 Primary Antibody Cyclin D1 (Polyclonalantibody) Wuhan Sanying 26939-1-AP WB 1:10000 Primary Antibody p-IκBα(Phospho-IκBAlpha, Ser32 / 36,Recombinant monoclonalantibody) Wuhan Sanying 82349-1-RR WB 1:2000 Primary Antibody IκBα (Polyclonalantibody) Wuhan Sanying 10268-1-AP WB 1:10000 Primary Antibody p-P65(Phospho-NF-κB p65,Ser468, Recombinant monoclonal antibody) Wuhan Sanying 82335-1-RR WB 1:5000 Primary Antibody P65 (NF-κB p65Recombinant monoclonalantibody) Wuhan Sanying 80979-1-RR WB 1:20000 Primary Antibody β-actin(Beta ActinMonoclonal antibody) Wuhan Sanying 66009-1-Ig WB 1:100000 isotype control Rabbit IgG control Wuhan Sanying 30000-0-AP Co-IP control; IP: 0.5–4.0 μg / 1.0–3.0 mg total protein Secondary antibody Goat Anti-Rabbit IgG HRP Abmart M21007 IP-WB, 1:5000 Secondary antibody CoraLite® Plus 594-GoatAnti-Rabbit (H+L) Wuhan Sanying RGAR004 IF, 1:500 Secondary antibody CoraLite® Plus 488-Goat Anti-Mouse (H+L) Wuhan Sanying RGAM002 IF, 1:500 Secondary antibody HRP-Goat Anti-Rabbit (H+L) Wuhan Sanying RGAR001 WB, 1:5000 Secondary antibody HRP-Goat Anti-Mouse (H+L) Wuhan Sanying RGAM001 WB, 1:5000 Main reagents DMEM culture medium Dalian Meilun MA0212 Cell culture Main reagents fetal bovine serum Ikosai FSP500 Cell culture Main reagents Penicillin-Streptomycin-Amphotericin B Mixed Solution Solarborg P7630 Cell culture Main reagents trypsin Dalian Meilun PWL059 Cellular digestion Main reagents Rapid sealing liquid Yamei PS108P WB closed Main reagents Universal antibody diluent Yamei PS119L Primary / Second Antibody Dilution Main reagents BCA Protein Concentration Kit Yamei ZJ102 Protein quantification Main reagents RIPA pyrolysis fluid Solarborg R0010 protein cleavage Main reagents Mixture of protease and phosphatase inhibitors Yamei GRF103 protein protection Main reagents TSA (Trichostatin A) MCE HY-15144 Maintaining Khib modification of protein Main reagents NAM (Nicotinamide) MCE HY-B0150 Maintaining Khib modification of protein Main reagents Trizol Cisco AC0101-B RNA extraction Main reagents Reverse transcription kit Novizan R323-01 cDNA Synthesis Main reagents Real-time PCR kit Yisheng 11201ES03 / 08 / 60 qPCR Main reagents ECL developing reagent Dalian Meilun MA0186 WB development Main reagents PVDF membrane Millipore IPVH00005 WB transfer Main reagents SDS-PAGE adhesive (10%) Yamei PG112 WB electrophoresis Main reagents 10×Tris-glycine SDS-PAGE electrophoresis buffer Servicebio G2027-1L Electrophoresis buffer Main reagents 10× Ice-Free Rapid Transfer Buffer Servicebio G2154-1L Transfer buffer Main reagents Omni-EasyTW Instant Protein Loading Buffer (Denaturing, Reducing, 5x) Yamei LT101 Protein loading buffer Main reagents Protein Marker Yamei WJ103 Protein molecular weight standards Main reagents 3% hydrogen peroxide solution Sigma 88597 Tissue staining related experiments Main reagents Reactive oxygen species (ROS) detection kit Dalian Meilun MA0219 Cellular ROS detection Main reagents Frozen Section Reactive Oxygen Species Staining Kit Solarborg G4817 ROS detection of frozen sections Main reagents N-acetylcysteine ​​(NAC) Abmole M5385 ROS clearance intervention Main reagents Protein A / G MagneticBeads MCE HY-K0202 Co-IP Main reagents EdU-647 Cell Proliferation Detection Kit Yamei CX004 EdU proliferation experiment Main reagents Tribromoethanol Dalian Meilun MB2548-1 Animal anesthesia Main reagents tert-amyl alcohol McLean A800283 Animal anesthesia 2. Experimental Methods 1.1 Cell culture, passage, and plating VSMCs were cultured in DMEM complete medium containing 10% fetal bovine serum and a 1% penicillin-streptomycin-amphoteric B mixture at 37°C and 5% CO2. After cell resuscitation, the cells were transferred to culture dishes for further culture. When cell confluence reached 80%–90%, the cells were passaged. After discarding the old medium, the cells were washed 1–2 times with PBS, digested with trypsin for about 3 minutes, and after the cells became rounded and partially detached, the digestion was stopped by adding complete medium. The cells were gently pipetted to form a single-cell suspension and then passaged at a 1:2 ratio. Cells were seeded into plates according to different experimental needs: approximately 2 × 10⁶ cells per well in a 6-well plate. 5 For each well in a 12-well plate, approximately 1 × 10^5 cells are seeded. The remaining wells are adjusted according to experimental requirements.

[0027] 1.2 Construction and cell transduction of PRDX2 lentiviral vector: This invention commissioned Obio Technology Co., Ltd. to construct and package recombinant lentiviruses with Flag tags for overexpressing PRDX2 wild-type (WT) and its modified mutants, respectively. Based on previous Khib research literature, the K10T mutant was used as a Khib functional mimic mutant at the PRDX2-K10 site, and the K10R mutant as a functional mimic mutant in the unmodified state at this site. Cells in the logarithmic growth phase were plated and infected with lentiviruses at a multiplicity of infection (MOI) of 40, with polybrene added simultaneously to enhance infection efficiency. After 24 hours of infection, the medium was replaced with fresh complete medium. Subsequently, selection was performed using medium containing puromycin (1 μg / mL) to obtain stable cell lines expressing PRDX2-WT, K10T, and K10R. The expression efficiency of all stable transfected cells was confirmed by Western blotting using Flag tags, and they were then used for subsequent functional and mechanistic experiments.

[0028] 1.3 PDGF-BB stimulation and drug intervention: To establish the optimal experimental conditions for inducing phenotypic transformation in human primary vascular smooth muscle cells (HA-VSMCs), preliminary experiments were conducted using concentration and time gradients of platelet-derived growth factor (PDGF-BB). HA-VSMCs were treated with different concentrations (5, 10, 20, 40 ng / mL) of PDGF-BB for 24 h, or with 40 ng / mL of PDGF-BB for different durations (6, 12, 24, 48 h). Changes in pan-2-hydroxyisobutyrylation (Pan-Khib) modification levels and the protein expression of the contractile marker α-SMA were detected to determine the optimal intervention strategy. Based on the preliminary gradient validation results, a final concentration of 40 ng / mL of PDGF-BB was used to treat HA-VSMCs for 24 h to construct a stable in vivo phenotypic transformation and pathological damage model. Based on the stable cell lines, the cells were divided into the following intervention groups according to the experimental objectives: blank control group (NC), PDGF-BB model group, PDGF-BB+PRDX2+WT group, PDGF-BB+K10T group (Khib functional mimic mutant), and PDGF-BB+K10R group (unmodified functional mimic mutant). Furthermore, to further verify the key role of ROS in this pathological process and protein modification regulation, an additional oxidative stress intervention was introduced on top of the above groupings: cells were treated with 50 μmol / L hydrogen peroxide (H2O2) for 24 h to exogenously upregulate intracellular ROS levels; simultaneously, cells were treated with 5 mM of the ROS scavenger N-acetylcysteine ​​(NAC) for 24 h to specifically block oxidative stress signals and observe its rescue effect.

[0029] 1.4 Establishment of a rat carotid artery balloon injury model and local AAV9 transduction: This invention utilizes Sprague-Dawley (SD) rats to establish a carotid balloon injury-induced angiogenesis and intimal hyperplasia model. Prior to the experiment, rats were generally anesthetized by intraperitoneal injection of Avertin anesthetic solution (2.5% w / v, 300 mg / kg) prepared with tribromoethanol. After routine skin preparation and disinfection, the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were dissected and exposed through a midline incision in the neck. A small incision was made through the ECA, and a 2F Fogarty balloon catheter was inserted retrogradely into the CCA near the aortic arch (at the level of the omohyoid muscle). The balloon was inflated to 2 atm, and while maintaining pressure, the catheter was pulled back at a constant speed to the incision site. This process was repeated five times to rub the vessel wall, thus eroding endothelial cells and inducing smooth muscle layer damage.

[0030] All recombinant adeno-associated virus type 9 (AAV9) vectors used were constructed and packaged by ObioTechnology. All AAV9 vectors used for in vivo transduction carried the SM22α smooth muscle-specific promoter to achieve relatively specific expression in vascular smooth muscle cells. Considering the difficulty of carotid artery microsurgical modeling, potential postoperative vascular embolism, and natural animal attrition, the initial sample size for each group was set at 10 rats (initial n=10 / group) to ensure a sufficient number of effective specimens were obtained. Rats were randomly divided into 5 groups: sham-operated group, injured control group, PRDX2-WT group, PRDX2-K10T group (Khib functional mimic mutant), and PRDX2-K10R group (unmodified functional mimic mutant). After balloon injury, a microartery clamp was temporarily used to block the proximal end of the CCA and ICA, forming a relatively closed local vascular lumen. A titer of 2×10⁻⁶ was slowly injected into this closed lumen through the ECA incision. 11 The corresponding AAV9 viral solution from vg (vectorgenomes) was incubated in situ for 20 minutes to mediate sufficient transduction of the virus to the damaged vascular wall. After incubation, the proximal ECA was ligated, the arterial clamp was released to restore normal blood flow from CCA to ICA, and the wound was sutured layer by layer. In the Sham group, only vascular dissection was performed, without balloon injury or viral perfusion.

[0031] 1.5 Tissue sampling and section preparation: Rats were routinely fed post-surgery and observed for two months. After the observation period, individuals that died due to surgical complications or whose modeling quality was substandard were excluded. Five rats from each group (effective statistical quantity n=5 / group) were randomly selected and euthanized using overdose anesthesia. The damaged carotid artery tissue was rapidly excised, and after dissecting the surrounding connective tissue, it was divided into two parts: one part was directly embedded in OCT embedding medium and flash-frozen in liquid nitrogen to prepare frozen sections for in situ tissue reactive oxygen species (DHE) fluorescence detection and morphological staining such as HE and EVG; the other part was directly flash-frozen in liquid nitrogen and then stored in an ultra-low temperature freezer at -80℃ for subsequent molecular biological detection.

[0032] 1.6 Immunofluorescence staining (IF): In this invention, both human and rat vascular specimens were prepared using a fresh cryosection method. Cryosections stored at -80°C were removed, allowed to thaw at room temperature, and then air-dried for 20-30 minutes to prevent detachment. Subsequently, the sections were post-fixed using 4% paraformaldehyde at room temperature for 15 minutes. After fixation, the sections were washed three times with PBS (5 minutes each time). Non-specific sites were blocked for 1 hour at room temperature using a solution containing 5% BSA. The blocking solution was removed, and appropriate concentrations of specific primary antibodies (Pan-Khib, 1:50; α-SMA, 1:1000) were added, and the sections were incubated overnight at 4°C in a humidified chamber. The next day, the sections were thoroughly washed with PBS, and the corresponding fluorescently labeled secondary antibodies were added and incubated at room temperature in the dark for 1 hour. After washing again, the cell nuclei were counterstained using a DAPI-containing anti-fluorescence quenching mounting medium, and the sections were mounted. Image acquisition was performed using an inverted fluorescence microscope, and the average fluorescence intensity of the target area was semi-quantitatively analyzed using ImageJ software.

[0033] 1.7 Western blot (WB) detection: Cell and vascular tissue samples were collected from each intervention group and lysed thoroughly on ice using RIPA lysis buffer pre-mixed with protease and phosphatase inhibitors. In the Pan-Khib detection experiment, to effectively inhibit demodification enzyme activity and maintain the true in vivo protein Khib modification state, protease inhibitors and deacetylase inhibitors nicotinamide (NAM, final concentration 50 mmol / L) and trachomatis A (TSA, final concentration 3 μmol / L) were additionally added to the lysis system. The samples were then centrifuged at 12000 r / min for 15 min at 4 °C, and the supernatant was collected. Total protein was accurately quantified using a BCA protein assay kit. Equal masses of protein samples were thoroughly mixed with corresponding volumes of loading buffer and denatured at 95 °C for 10 min. Equal volumes of denatured protein samples were separated by SDS-PAGE gel electrophoresis and then transferred to PVDF membranes under constant current. The PVDF membranes were then blocked in rapid blocking buffer at room temperature for 15–30 min to block non-specific binding. After washing, the corresponding primary antibodies (Pan-Khib, PRDX2, Flag, α-SMA, MMP2, MMP9, PCNA, Cyclin D1, p-IκBα, IκBα, p-P65, P65, and internal control β-actin; antibody sources and dilution ratios are detailed in Table 1) were added according to predetermined ratios and incubated overnight at 4°C on a shaker. The next day, the PVDF membrane was washed three times with TBST buffer for 10 min each time; then, the corresponding horseradish peroxidase (HRP)-labeled secondary antibody was added and incubated at room temperature for 1 h. After washing the membrane thoroughly with TBST again, ultrasensitive ECL chemiluminescence solution was added for chemiluminescence development, and the band signals were captured using a fully automated imaging system. Finally, the gray values ​​of the obtained protein bands were semi-quantitatively analyzed using ImageJ software, and the relative expression level of the target protein was characterized by the ratio of the gray values ​​of the target protein to the internal control protein (β-actin).

[0034] 1.8 Co-immunoprecipitation (Co-IP): To assess the Khib level of the target protein PRDX2, this invention employs Protein A / G magnetic beads for Co-IP experiments. Cells were washed twice with pre-cooled PBS and then lysed with RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors. To effectively inhibit demodification enzyme activity and maintain the true Khib state of the protein in vivo, protease inhibitors, deacetylase inhibitors nicotinamide (NAM, final concentration 50 mmol / L), and trachomatis A (TSA, final concentration 3 μmol / L) were strictly added to the lysis system. Samples were thoroughly lysed on ice for 30 min (vortexed every 10 min), followed by centrifugation at 12,000 × g for 15 min at 4 °C. The supernatant was collected, and total protein was accurately quantified using the BCA method.

[0035] In the Co-IP process, 100 μg of total protein from each sample group was retained as a whole-cell lysis buffer control (Input). For the IP experimental group, 1 mg of total protein was taken and thoroughly mixed with 30 μL of pre-washed Protein A / G magnetic beads and 2.5 μL of PRDX2 antibody, and incubated overnight at 4°C on a rotary mixer. The next day, the sample tubes were placed on a magnetic rack for magnetic separation, the supernatant was discarded, and the magnetic beads were resuspended and washed 5 times with pre-cooled lysis buffer (each wash at 4°C for about 10 min) to thoroughly wash away non-specifically bound proteins. After washing, 30 μL of 1× protein denaturation loading buffer was added directly to the magnetic bead precipitate, and the mixture was heated in a 95°C metal bath for 10 min to elute and denature the antigen-antibody complex. Finally, the eluent was collected for Western blotting, and the modification level of the enriched target protein PRDX2 was analyzed using modified pan-Khib antibody.

[0036] 1.9 RNA extraction and real-time quantitative PCR (RT-qPCR): Total RNA was extracted from cells or vascular tissues in each intervention group using Trizol reagent. The concentration and purity of the RNA were determined using a micro-ultraviolet spectrophotometer to ensure the A260 / A280 absorbance ratio was between 1.8 and 2.0. Subsequently, an equal volume of 1 μg of qualified total RNA was taken and reverse transcribed into single-stranded cDNA strictly according to the reverse transcription kit instructions.

[0037] Amplification and detection were performed using a SYBR Green real-time quantitative PCR mixed enzyme on an Agilent real-time quantitative PCR instrument. In this invention, in vitro human cell experiments primarily detected the mRNA expression levels of ICAM1, CCL2, and IL6 (using human GAPDH as an internal control); in vivo rat experiments detected the corresponding mRNA expression levels of ICAM1, CCL2, and IL6 (using rat GAPDH as an internal control). Relevant specific primers were designed and synthesized by Sangon Biotech. After the amplification reaction, the specificity of the PCR products was verified by analyzing the melting curve, and standard 2... -△△Ct The relative transcriptional expression levels of each target gene were calculated.

[0038] 2.10 Detection of intracellular ROS levels: The accumulation level of intracellular ROS in each group of cells was detected using the DCFH-DA fluorescent probe kit (Table 1). After cells were treated according to the predetermined groups, an appropriate amount of DCFH-DA working solution was added according to the instructions, and the cells were incubated at 37°C in the dark. After washing three times with PBS to remove free probes outside the cells, the cells were immediately observed under an inverted fluorescence microscope, and images were randomly acquired. The intracellular ROS concentration was directly proportional to the intensity of the excited green fluorescence. The mean fluorescence intensity (MFI) of each group of cells was measured using ImageJ software for semi-quantitative analysis.

[0039] 2.11 EdU cell proliferation experiment: The proliferation of cells in each group was detected using the EdU-647 cell proliferation assay kit (Table 1). After treatment according to the predetermined groups, cells were incubated with EdU working solution at 37°C for 2 hours. Subsequently, cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100. Click reaction incubation in the dark and Hoechst nuclear counterstaining were performed strictly according to the kit instructions to specifically label proliferating cells. Multiple independent fields of view were randomly selected for image acquisition under an inverted fluorescence microscope. ImageJ software was used to count the cells, calculating the percentage of EdU-positive cells to the total cell count, thus quantitatively reflecting the cell proliferation capacity.

[0040] 2.12 Transwell migration experiment: To evaluate the chemotactic migration ability of vascular smooth muscle cells under different intervention conditions, this invention uses Transwell chambers with an 8.0 μm pore size for in vitro experiments. Cells in each group were treated according to predetermined conditions, routinely digested, and resuspended in serum-free medium to adjust cell density. An equal volume of 200 μL of single-cell suspension was evenly seeded into the upper chamber of the Transwell chamber. Simultaneously, 500 μL of complete culture medium was added to the lower chamber as a chemotactic inducer.

[0041] The culture plates were incubated at 37°C with 5% CO2 for 12 h and 24 h respectively. After incubation, the chambers were removed, and cells that had not migrated were gently wiped off the upper surface of the filter membrane with sterile cotton swabs. The chambers were fixed in 4% paraformaldehyde at room temperature for 15 min, and then immersed in 0.1% crystal violet solution for staining at room temperature for 20 min. After thorough rinsing with PBS, the chambers were observed under an inverted microscope. At least three non-overlapping fields of view were randomly selected from each chamber for image acquisition, and the number of cells that had crossed to the lower surface of the filter membrane was counted using ImageJ software. The average number of cells that had perforated the membrane was used as a quantitative indicator to evaluate the migration potential of cells in each group.

[0042] 2.13 Scratch healing experiment: The two-dimensional migration ability of vascular smooth muscle cells in each group was assessed using the in vitro scratch healing assay. Cells in the logarithmic growth phase were seeded uniformly at an appropriate density in 6-well culture plates. When the cells adhered and grew to approximately 90%–100% confluence to form a monolayer of cells, the medium was replaced with serum-free medium and starved for 12 hours to synchronize the cell cycle and eliminate interference from cell proliferation on the migration results.

[0043] After starvation, a straight cell scratch was made vertically and at a constant speed using a sterile 200 μL pipette tip at the bottom of the well plate. The old culture medium was then discarded, and the cells were gently washed three times with sterile PBS solution to thoroughly remove any detached and suspended cells from the scratch edges. After washing, 1% low-serum culture medium containing the appropriate intervention drug was added to each well for continued culture. Representative images were acquired at the same fixed position under an inverted microscope at both the scratch initiation point and the predetermined endpoint after treatment. Finally, the images were imported into ImageJ software to precisely delineate and measure the area of ​​the cell-free region. The wound closure rate (%) was calculated using the formula: Wound Closure Rate (%) = [(0h scratch area - endpoint scratch area) / 0h scratch area] × 100%, thus quantitatively evaluating the dynamic migration ability of cells in each group.

[0044] 2.14 Khib modification proteomics and analysis: Total protein was extracted from healthy controls and ISR vascular tissue (3 μM TSA and 50 mM NAM were strictly added to the lysis system to maintain Khib modification). After quantification with BCA and digestion with trypsin, Khib-modified peptides were enriched using specific affinity resin and desalted using a C18 column. Subsequently, high-throughput mass spectrometry detection of modified peptides and total protein was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS, timsTOFPro2). The raw mass spectrometry data were imported into MaxQuant (v1.6.15.0) software for library search and comparison. Under strict control of the false positive rate (FDR < 1%), significantly differentially expressed proteins and key modification sites were screened based on a fold change > 1.5 and p < 0.05. All raw mass spectrometry data of proteomics and Khib modification proteomics have been uploaded to the ProteomeXchange Consortium (dataset number: PXD062384) via the PRIDE database; the identified related proteins and Khib modification spectra have been stored in the MS-viewer database (search codes: "4xxtqp3gqg" and "kc21vnele4").

[0045] 2.15 Bioinformatics screening of the core target PRDX2 and its key modification sites: To accurately isolate interference from changes in protein expression alone and focus on the independent regulatory role of Khib modification in disease progression, this invention employs a rigorous omics-based cross-screening strategy. First, modification-based and total proteomics data are jointly compared to specifically identify candidate proteins that show no significant difference in total protein expression levels between disease (ISR) and health (HEALTH) states, but exhibit significant downregulation of Khib modification levels (screening criterion: modification level difference P < 0.05).

[0046] To further pinpoint the core molecule, Gene Ontology (GO) functional enrichment analysis was performed on proteins corresponding to the most significantly differentially modified sites (DISEASE / HEALTH Ratio < 0.4) in the aforementioned candidate pool. The results showed that these differentially modified proteins were highly enriched in redox biological functions. Combining literature and functional analysis, peroxiredoxin-2 (PRDX2), as a key intracellular antioxidant and reductive regulatory enzyme, came into the scope of this invention. Omics data showed that, with no change in total expression level, the K10 site of PRDX2 showed a significant downregulation of Khib modification in the disease group (DISEASE / HEALTH Ratio = 0.0707), ranking third among all proteins with unchanged expression but downregulated modification sites. Therefore, it was ultimately identified as the core target for subsequent molecular mechanism investigation.

[0047] Target Validation and Conservation Analysis: Based on the above omics screening results, the UniProt database was used to annotate the domains of the PRDX2 protein, and cross-species amino acid sequence homology alignment was performed to assess the high conservation of the K10 site during evolution. Simultaneously, combining the previously established rat carotid balloon injury model and in vitro PDGF-BB stimulation model, Co-IP combined with Western blotting was used to conduct dual experimental validation of the dynamic downregulation of Khib modification levels of PRDX2 under in vitro and in vivo pathological stress conditions.

[0048] 2.16 Transcriptome sequencing and bioinformatics analysis: Human VSMCs stably overexpressing PRDX2-K10T (Khib functional mimic mutant) and PRDX2-K10R (unmodified functional mimic mutant) were selected. Total RNA was extracted after stimulation with 40 ng / mL PDGF-BB for 24 h (3 biological replicates per group, 6 samples in total). mRNA with polyA tails was specifically enriched using oligo-dT magnetic beads, and the target fragments were randomly fragmented into 200–300 nt fragments by heating. cDNA was synthesized by reverse transcription using the fragmented mRNA as a template, followed by end repair, 3' A-base addition, and sequencing adapter ligation. Sequencing libraries were constructed by PCR amplification and fragment size selection (products concentrated in the 300–400 bp range). After library quality control, high-throughput sequencing was performed using the Illumina NovaSeq 6000 platform.

[0049] For the raw reads obtained from the sequencing process, the Q20 and Q30 ratios were first assessed using the FastQC (v0.11.9) program, and adapter sequences, short sequences, and low-quality reads were removed using the fastp (v0.23.2) software. The obtained high-quality clean data was aligned to the human reference genome (hg38) using the HISAT2 (v2.1.0) program, with only high-confidence reads (MAPQ) ≥ 30 retained for subsequent calculations. Transcript splicing and gene expression level quantification were performed using StringTie (v2.1.4) software combined with the Ensembl v101 annotation file. Subsequently, differentially expressed gene analysis was performed using the edgeR (v3.28.1) program, with statistical screening thresholds strictly set to p-value < 0.05 and fold change > 1.5 or < 0.66. Finally, the clusterProfiler package in R was used to perform gene ontology (GO) annotation and KEGG signaling pathway enrichment analysis on the obtained differentially expressed genes in order to systematically elucidate the downstream transcriptional regulation mechanism mediated by the alteration of the PRDX2-K10 site modification status.

[0050] 2.17 HE, EVG, and DHE staining: HE staining: Frozen sections of rat carotid arteries (approximately 5 μm thick) stored at -80℃ were removed, thawed to room temperature, and air-dried. They were then fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the sections were immersed in hematoxylin staining solution for 3–5 min to stain the nuclei. After rinsing with tap water, they were differentiated with 1% hydrochloric acid alcohol for a few seconds, followed by blueing with a blueing solution or running water. The sections were then immersed in eosin staining solution to stain the cytoplasm for 1–2 min. Finally, they were dehydrated stepwise with a gradient of alcohols (70%, 80%, 90%, 95%, 100%), cleared with xylene, and mounted with neutral resin.

[0051] EVG staining (elastic fiber staining): After rewarming and fixing the sections as above, immerse them in freshly prepared Verhoeff staining solution and incubate at room temperature for 15–30 min, then rinse with running water. Differentiation is then performed using 2% ferric chloride solution until the elastic fibers appear as a clear purplish-black color with a grayish-white background under an optical microscope. After thorough rinsing with tap water, counterstain with Van Gieson (VG) staining solution for 1–2 min. Quickly dehydrate with 95% ethanol and anhydrous ethanol, clear with xylene, and mount with neutral resin.

[0052] DHE staining (in situ ROS detection): To accurately assess the level of oxidative stress in the local blood vessel wall under pathological conditions, fresh, unfixed frozen sections were strictly used for detection. After drying and thawing at room temperature for 3-5 min, the sections were treated with 1× washing buffer provided with the kit for 10 min at room temperature. After aspirating the liquid, diluted DHE working solution was added to completely cover the tissue, and the sections were incubated at room temperature in the dark for 1 h. After washing three times with PBS, DAPI staining solution was added to counterstain the cell nuclei for 10 min at room temperature in the dark. After washing again, the sections were mounted with anti-fluorescence attenuation mounting medium.

[0053] Image Acquisition and Quantitative Analysis: All stained sections were imaged using an inverted microscope. HE and EVG sections were acquired under bright-field imaging to obtain complete cross-sectional images of the target vessels; DHE sections were acquired under fluorescence imaging. Images were uniformly imported into ImageJ software for quantitative analysis: For HE and EVG morphological images, the contours were manually delineated along the luminal endothelial margin and the peripheral boundary of the vessel. The luminal area (in units of 10³ μm²) and the total vessel area (Vessel area) were accurately measured, and the ratio of luminal area to total vessel area (Luminal Area / VesselArea Ratio, %) was calculated to intuitively and objectively assess the degree of luminal occlusion and pathological remodeling caused by neointimal hyperplasia. For DHE images, the mean fluorescence intensity of the target area was measured to semi-quantitatively analyze the local reactive oxygen species (ROS) accumulation level.

[0054] 2.18 Statistical Analysis: The extraction of raw quantitative data such as blot band grayscale, histological area, and in situ fluorescence intensity involved in this invention was performed using ImageJ (v1.53) software. All statistical calculations and the creation of statistical charts were performed using GraphPadPrism (v10.1.2) software.

[0055] Experimental data were derived from at least three independent biological replicates. Quantitative data are expressed as mean ± standard deviation (x ± s). Two-tailed unpaired t-tests were used for comparisons between two groups; Welch correction was applied when variances were unequal. One-way ANOVA was used for comparisons among multiple groups; after statistical significance was established, Tukey's post-hoc test was used for pairwise comparisons. A p-value < 0.05 was considered statistically significant, and was indicated with an asterisk in the statistical tables (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

[0056] 3. Experimental results; 1. The overall level of Pan-Khib in human ISR blood vessels and damaged rat blood vessels was significantly decreased: To clarify the expression changes of 2-hydroxyisobutyrylation (Khib) modification during restenosis after vascular injury, the overall level of pan-2-hydroxyisobutyrylation (Pan-Khib) was first examined in human in-stent restenosis (ISR) vessel specimens and rat carotid balloon-injured vessels. Immunofluorescence staining results showed that the Pan-Khib fluorescence signal in human ISR vessels was significantly weakened compared with healthy control vessels (AB in Figure 1). Further Western blot (WB) protein quantification analysis confirmed that the relative expression level of Pan-Khib protein in human ISR specimens was significantly lower than that in the healthy control group (CD in Figure 1). Subsequently, we validated this phenomenon in a rat carotid balloon injury model. Immunofluorescence results also showed that the Pan-Khib fluorescence signal in rat injured vessels was significantly weakened compared with the sham-operated group (EF in Figure 1). At the same time, WB detection results of rat tissue samples also showed that the expression of Pan-Khib protein in injured vessels was significantly reduced compared with the sham-operated group (GH in Figure 1). The above results consistently suggest that there is a relatively stable and significant state of low Khib modification expression during restenosis after vascular injury.

[0057] 2. Khib modification proteomics combined with proteomics screening identified PRDX2 K10 as a key differentially modified site: To screen key Khib-modified proteins and their sites of action mediating restenosis after vascular injury, this invention conducted in-depth analysis combining Khib modification proteomics and proteomics. In a comparison between the disease and healthy groups, the overall number of differentially modified proteins and sites was first counted (Figure 2A). Volcano plots further visually demonstrated the significant differential changes in Khib modification levels of various proteins during ISR (Figure 2B). To further pinpoint core molecules, Gene Ontology (GO) functional enrichment analysis was performed on the candidate set with the most significant modification differences (disease / health ratio < 0.4). The results showed that these differentially modified proteins were highly enriched in redox biological functions (…). Figure 2 (C). Based on literature review and functional analysis, peroxidase 2 (PRDX2), a key intracellular antioxidant and reductive regulatory enzyme, came into focus in this study. Quantitative omics data showed that, while the total expression level of PRDX2 remained unchanged, the K10 site showed a significant downregulation of Khib modification in the disease group (disease / health ratio = 0.0707, ranking third among all proteins with unchanged total expression but downregulated modification). Therefore, it was identified as the core target for subsequent investigation of its molecular mechanisms.

[0058] Based on the above omics screening results, the PRDX2 protein was annotated using the UniProt database, precisely locating the K10 site in the sequence (Figure 2, D). Cross-species amino acid sequence homology alignment confirmed that this K10 site exhibits high conservation throughout human, primate, rat, and mouse evolution (Figure 2, E). Subsequently, tandem mass spectrometry (MS / MS) analysis precisely confirmed the presence of the Khib-modified peptide at the PRDX2 K10 site (Figure 2, F). These results suggest that the highly conserved PRDX2-K10 site may be a key Khib modification site in the restenosis process following vascular injury.

[0059] 3. Both vascular injury and PDGF-BB stimulation are accompanied by a decrease in the Khib modification level of PRDX2: To further validate the changes in PRDX2 Khib modification suggested by omics screening, the Khib modification level of PRDX2 in a rat carotid artery balloon injury model was detected by Co-IP combined with Western blotting. The results showed that, compared with the uninjured control group, the Khib signal of PRDX2 Co-IP product in the injured vascular tissue was significantly weakened, suggesting that the Khib modification level of PRDX2 decreased under vascular injury conditions (Figure 3A).

[0060] Subsequently, to clarify the stimulation conditions in vitro, concentration gradient and time gradient experiments were conducted. Western blotting results showed that after 24 h of treatment with different concentrations of PDGF-BB, the overall pan-2-hydroxyisobutyrylylation (Pan-Khib) level in VSMCs generally decreased with increasing stimulation concentration, while the expression of the contractile phenotypic marker α-SMA also gradually decreased (Figure 3B). Further time gradient experiments showed that under 40 ng / mL PDGF-BB stimulation, the Pan-Khib level and α-SMA expression in VSMCs further decreased with prolonged treatment time (Figure 3C). Based on these results, subsequent in vitro experiments used 40 ng / mL PDGF-BB stimulation for 24 h as the cell model condition.

[0061] Building upon this, Co-IP combined with Western blotting was used to detect the Khib modification level of PRDX2 in VSMCs. The results showed that, compared with the unstimulated control group, the Khib signal in the PRDX2 Co-IP product was significantly decreased after PDGF-BB stimulation, while the total PRDX2 protein level in the input sample showed no significant change (Figure 3D). These results indicate that vascular injury and PDGF-BB stimulation are both accompanied by a decrease in the Khib modification level of PRDX2, and are related to the overall decrease in Khib levels in VSMCs and the phenotypic transformation process.

[0062] 4. Simulating and maintaining the Khib state at the PRDX2 K10 site can significantly reduce ROS accumulation in VSMCs and inhibit the inflammatory response: To further explore the specific function of Khib modification at the PRDX2 K10 site in the phenotypic regulation of VSMCs, lentiviral overexpression vectors targeting the expression of wild-type PRDX2 (WT), a Khib-mimicking mutant (K10T), and a Khib-free mutant (K10R) were successfully constructed (Figure 4A). Western blot analysis confirmed that all recombinant lentiviruses were successfully transduced into VSMCs, and the flag bands indicated that the expression levels of the three groups of exogenous proteins (WT, K10T, and K10R) were generally comparable (Figure 4B). Under PDGF-BB stimulation, the dynamic changes in intracellular reactive oxygen species (ROS) levels were assessed using the DCFH-DA fluorescent probe. The results showed that PDGF-BB significantly induced abnormal ROS accumulation in VSMCs; although overexpression of PRDX2-WT could alleviate ROS production to some extent, the Khib-mimicking K10T mutant exhibited more significant antioxidant stress efficacy. In contrast, the K10R mutant, which blocked the modification at this site, showed a weakened protective effect (CD in Figure 4). Further RT-qPCR analysis showed that PDGF-BB stimulation significantly upregulated the mRNA transcription levels of pro-inflammatory cytokines IL-6, MCP-1, and ICAM-1. Consistent with the changes in ROS, both WT and K10T effectively downregulated the expression of these inflammatory cytokines, with K10T showing the most significant inhibitory effect; while the anti-inflammatory effect of the K10R group was significantly weakened (EG in Figure 4). These results indicate that simulating the Khib modification state at the PRDX2 K10 site can effectively suppress the inflammatory response of VSMCs by clearing intracellular ROS accumulation.

[0063] 5. The Khib state at the PRDX2 K10 site inhibits the expression of VSMC proliferation and migration-related proteins: To further evaluate the effect of PRDX2 K10 site modification on abnormal phenotypic transformation in VSMCs at the molecular level, Western blotting (WB) was used to detect changes in the expression of core marker proteins related to cell proliferation and migration. Representative WB images (Figure 5A) and corresponding quantitative analysis results showed that after PDGF-BB stimulation, the expression levels of migration-related proteins MMP9 (Figure 5B) and MMP2 (Figure 5C), as well as proliferation-related proteins PCNA (Figure 5D) and Cyclin D1 (Figure 5E), were significantly upregulated in VSMCs. After gene intervention, overexpression of PRDX2-WT partially reversed the abnormal increase in these proteins; while the Khib functional mimic mutant K10T showed a more significant inhibitory effect, significantly downregulating the expression of these proliferation and migration markers. In contrast, the unmodified functional mimic mutant K10R showed a significantly weaker inhibitory effect on the abnormal increase in these proteins. These results indicate that simulating the Khib modification state at the PRDX2 K10 site can effectively inhibit the activation of molecular programs related to VSMC proliferation and migration under pathological stimulation.

[0064] 6. Simulated maintenance of the Khib state at the PRDX2 K10 site can significantly inhibit the proliferation and migration ability of VSMCs: To further verify the regulatory role of PRDX2 K10 site Khib modification status on phenotype transformation of VSMCs at the cellular functional level, EdU cell proliferation, Transwell cell migration, and scratch healing experiments were conducted. First, representative images visually demonstrate the morphological phenotypic differences among the experimental groups in EdU fluorescence staining (Figure 6A), Transwell (Figure 6B), and scratch healing (Figure 6C).

[0065] Subsequent quantitative analysis further confirmed the above observations. Regarding cell proliferation, the EdU-positive cell ratio results (Figure 6, D) showed that PDGF-BB stimulation significantly promoted VSMC proliferation; PRDX2-WT overexpression partially reversed this change, while K10T showed the most significant inhibitory effect; in contrast, the inhibitory effect of the K10R group was significantly weakened. Regarding cell migration, the Transwell assay results at 12 h (Figure 6, E) and 24 h (Figure 6, F) indicated that PDGF-BB induced a large number of cell migrations; both WT and K10T reduced the number of transmembrane cells, with K10T having a more significant effect, while the anti-migration effect of the K10R group was weaker. Quantitative analysis of the scratch healing assay (Figure 6, G) yielded consistent results: PDGF-BB significantly accelerated wound closure, both WT and K10T reduced the closure rate, while K10R had a relatively limited intervention effect on abnormal migration. The above multidimensional functional results indicate that simulating the Khib functional state of the PRDX2 K10 site can inhibit the proliferation and migration of VSMCs under pathological stimulation.

[0066] 7. Exogenous ROS can partially reverse the protective effect of the K10T mutant against the abnormal phenotype of VSMCs: To clarify the specific mechanism by which reactive oxygen species (ROS) play a role in the phenotype regulation of VSMCs mediated by the PRDX2 K10 site, 50 μmol / L hydrogen peroxide (H2O2) was introduced as an exogenous ROS donor for 24 hours as an intervention in the context of K10T mutant (mimicking the Khib modified state) overexpression. Representative images of the DCFH-DA fluorescent probe (Figure 7A) and corresponding quantitative analysis (Figure 7B) confirmed that K10T could significantly clear intracellular ROS accumulation induced by PDGF-BB; and after H2O2 stimulation, the fluorescence intensity of the previously inhibited ROS in the cells increased again.

[0067] Following the recovery of intracellular oxidative stress, morphological and functional changes in cells were further assessed. Representative EdU-stained images (Fig. 7C) and representative scratch wound healing images (Fig. 7D) visually demonstrate the H2O2-mediated reactivation trend of exogenous ROS in cell proliferation and migration. Subsequent detailed quantitative analysis further supports that the addition of H2O2 partially offset the downregulation effect of the K10T mutant on the proportion of EdU-positive proliferating cells (Fig. 7E), and also significantly restored the scratch closure rate originally blocked by K10T (Fig. 7F). The results of the above recovery experiments suggest that the phenotypic protective effect of simulating the Khib modification state at the PRDX2 K10 site on VSMCs depends, at least to a large extent, on its effective clearance of intracellular ROS accumulation.

[0068] 8. The ROS scavenger NAC can partially reverse the high ROS and abnormal proliferation and migration phenotype mediated by the K10R mutant: To further confirm the core mediating role of ROS in this molecular mechanism from a reverse perspective, the ROS scavenger N-acetylcysteine ​​(NAC) was introduced into VSMCs overexpressing the K10R mutant (blocking the Khib modification state). Representative images of the DCFH-DA fluorescent probe (Figure 8A) and corresponding quantitative analysis (Figure 8B) showed that K10R group cells maintained a high level of endogenous ROS accumulation under PDGF-BB stimulation; while after NAC treatment, the abnormally high intracellular ROS fluorescence intensity was significantly inhibited and decreased substantially.

[0069] Following the relief of intracellular oxidative stress, the corresponding changes in cell phenotype were further observed. Representative EdU staining images (Figure 8, C) and representative scratch healing images (Figure 8, D) visually demonstrate the blocking effect of NAC on excessive cell proliferation and migration. Subsequent detailed quantitative analysis confirmed that the addition of NAC significantly reduced the abnormally elevated proportion of EdU-positive cells under K10R conditions (Figure 8, E) and effectively slowed down the stimulated scratch closure rate (Figure 8, F). These results of bidirectional intervention corroborate each other, suggesting that the regulation of VSMC phenotype by the Khib state at the PRDX2 K10 site depends at least in part on the maintenance of intracellular ROS homeostasis.

[0070] 9. Transcriptome sequencing combined with Western blotting reveals that the Khib status at the PRDX2 K10 site regulates the inflammatory response through the ROS / NF-κB axis: To further explore the specific downstream molecular mechanisms by which Khib modification at the PRDX2 K10 site regulates the phenotype of VSMCs, transcriptome sequencing (RNA-seq) analysis was performed on stable K10T (modified group) and K10R (demodified group) cell lines. Volcano plots (Figure 9A) and cluster heatmaps (Figure 9B) visually revealed significant differential gene expression profiles between the two groups. Subsequent KEGG pathway enrichment analysis indicated that the differentially expressed genes were mainly enriched in biological pathways closely related to VSMC inflammation, proliferation, and migration, such as the TNF signaling pathway, IL-17 signaling pathway, chemokine signaling pathway, and NF-κB signaling pathway (Figure 9C). Gene set enrichment analysis (GSEA) further confirmed significant changes in NF-κB signaling pathway activity between the two groups (Figure 9D).

[0071] To validate the above omics analysis results at the protein level, we focused on examining the phosphorylation activation status of key NF-κB pathway proteins. Representative Western blotting images (Figure 9, E) and quantitative analysis confirmed that PDGF-BB stimulation significantly upregulated the expression levels of p-IκBα and p-P65 in VSMCs; while overexpression of PRDX2-WT and K10T both inhibited the phosphorylation of these proteins to varying degrees, with K10T showing better inhibitory effects than the WT group. More importantly, after introducing exogenous ROS (H2O2) intervention in the K10T mutant background, the previously inhibited p-IκBα (Figure 9, F) and p-P65 (…)… Figure 9 G) expression levels rebounded significantly again. Conversely, in the context of blocking Khib-modified K10R, representative WB ( Figure 9 The results of the bidirectional response experiment (H) and its quantitative analysis showed that treatment with the ROS scavenger NAC significantly downregulated the abnormally elevated phosphorylation activation levels of p-IκBα (I in Figure 9) and p-P65 (J in Figure 9). These bidirectional response results conclusively demonstrate that maintaining the Khib modification state at the PRDX2 K10 site can effectively scavenge endogenous ROS, thereby blocking the abnormal activation of the downstream NF-κB signaling pathway and ultimately exerting an inhibitory effect on VSMC inflammation, proliferation, and migration.

[0072] 10. AAV9-mediated PRDX2-K10T expression significantly reduced neointimal hyperplasia, oxidative stress, and inflammatory response after balloon injury in vivo. To further verify the biological effects of the in vitro mechanism at the in vivo level, an in vivo intervention was performed using a Sprague-Dawley (SD) rat carotid artery balloon injury model. Recombinant adeno-associated virus (AAV9) carrying the SM22α smooth muscle-specific promoter was locally perfused to specifically overexpress PRDX2-WT, K10T, and K10R in the target vessels. Representative images of the target vessels after surgery, stained with hematoxylin-eosin (HE) and elastic fiber (EVG) (Figure 10A), visually showed that the intima of the injured rats was highly thickened, and the lumen was severely narrowed. Compared with the simple injury group, AAV9-mediated overexpression of WT and K10T effectively inhibited abnormal neointimal proliferation and improved luminal narrowing; among them, the K10T group, which simulated the Khib-modified state, showed the most significant morphological rescue effect, while the protective effect of the K10R blocking modification group was significantly attenuated. Further morphological quantitative analysis confirmed the above observations: the absolute lumen area (Figure 10B) and the ratio of lumen area to total vascular area (Figure 10C) of rats in the K10T group showed the greatest degree of recovery, which was significantly better than that of the injury group and the K10R group.

[0073] While assessing the morphology of vascular remodeling, we further examined the changes in the local microenvironment of the damaged vessels. Representative images of dihydroethidium (DHE) fluorescence staining (Figure 10, D) and their quantitative analysis of relative fluorescence intensity (Figure 10, E) showed that balloon-induced endothelial injury induced severe accumulation of reactive oxygen species (ROS) in the vessel wall; while local transduction of K10T was able to more efficiently reduce the abnormal oxidative stress response of the vessel wall, and its ability to scavenge ROS was significantly better than that of the WT group and the K10R group. In addition, RT-qPCR detection results of the diseased vessel tissue confirmed that vascular injury significantly upregulated the expression levels of pro-inflammatory cytokines IL-6, MCP-1, and ICAM-1. Consistent with the trend of improvement in morphology and oxidative stress, both WT and K10T interventions effectively downregulated the transcription of these inflammatory factors, with the K10T group showing a more significant anti-inflammatory effect. Figure 10 The K10R group showed limited inhibitory effects on local inflammation. These in vivo animal experiments further demonstrate that maintaining the Khib modification state at the PRDX2 K10 site effectively inhibits vascular injury-induced local oxidative stress, inflammatory responses, and luminal stenosis in vivo.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Application of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of products that inhibit VSMC phenotypic transformation.

2. Application of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of products that inhibit the proliferation and / or migration of VSMCs.

3. Application of PRDX2-K10 site 2-hydroxyisobutyrylation modification in the preparation of drugs for preventing restenosis after vascular injury.

4. The application of PRDX2-K10 site 2-hydroxyisobutyrylated mimic in the preparation of products that inhibit VSMC phenotypic transformation, characterized in that, The PRDX2-K10 site 2-hydroxyisobutyrylation modification mimicry is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

5. The application of the PRDX2-K10 site 2-hydroxyisobutyrylated mimic in the preparation of products that inhibit the proliferation and / or migration of VSMCs, characterized in that, The PRDX2-K10 site 2-hydroxyisobutyrylation modification mimicry is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

6. The application of PRDX2-K10 site 2-hydroxyisobutyrylated mimicry in the preparation of drugs for preventing restenosis after vascular injury, characterized in that, The PRDX2-K10 site 2-hydroxyisobutyrylation modification mimicry is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

7. The application of a recombinant vector expressing a mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation in the preparation of products inhibiting VSMC phenotypic transformation, characterized in that, The PRDX2-K10 site 2-hydroxyisobutyrylation modification mimicry is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

8. The application of a recombinant vector expressing a mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation in the preparation of products that inhibit the proliferation and / or migration of VSMCs, characterized in that, The PRDX2-K10 site 2-hydroxyisobutyrylation modification mimicry is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

9. The application of a recombinant vector expressing a mimicry of PRDX2-K10 site 2-hydroxyisobutyrylation in the preparation of drugs for preventing restenosis after vascular injury, characterized in that, The PRDX2-K10 site 2-hydroxyisobutyrylation modification mimicry is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

10. A drug for preventing restenosis after vascular injury, characterized in that, The drugs for preventing restenosis after vascular injury include PRDX2-K10 site 2-hydroxyisobutyrylation modified mimics and / or recombinant vectors expressing PRDX2-K10 site 2-hydroxyisobutyrylation modified mimics; The PRDX2-K10 site 2-hydroxyisobutyrylation modification mimicry is obtained by mutating the 10th K lysine in the amino acid sequence of the PRDX2 protein to threonine T.

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