AgNP material characterization and Se sample preparation
AgNP of 20 nm sizes were obtained from US Research Nanomaterials, Inc. AgNP were dissolved and stored according to a previous method [19]. Before transmission electron microscopic examination (TEM) analysis, the stock suspension of AgNP was immersed in ultrapure water and underwent three rounds of ultrasonic oscillations at a low-temperature for a duration of 30 min.
To obtain 100 mM concentration of Se stock solutions, 0.1729 g of Se (Sigma, USA) was dissolved in 10 mL ultrapure water. Following this, the stock solution underwent dilution using the same medium, resulting in a 1 mM working solution, which was subsequently stored at -20℃.
Experimental animals and intervention
The Medical Laboratory Animal Center of Ningxia Medical University provided forty identical, male Sprague-Dawley (SD) rats weighing between 220-240g. In line with the established feeding standard, each rat was confined to standardized cages under a 12 h light/dark cycle, and exposed to standard temperature and relative humidity levels ranging from 54-59%. These animals were provided with a standard pellet diet and unrestricted access to water. In our study, all animal experiments were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals. The study received approval from the Laboratory Animal Ethical and Welfare Committee of Ningxia Medical University (IACUC-NYLAC-2022-202).
In order to examine the protective effect of Se on AgNP-induced damage to the endothelial cells of the aorta, the animals were randomly divided into 4 groups following a one-week acclimatization period: (1) Control group (Control): rats were administered 200 μL of saline solution via intratracheal instillation; (2) AgNP intervention group (AgNP): each rat was treated with 200 μL AgNP by tracheal perfusion with only once; (3) Selenium group (Se): sodium selenite dissolved in distilled water (0.2 mg/kg/day) was intraperitoneally administered for 7 days. (4) AgNP+Selenium group (AgNP+Se): each rat received 200 μL AgNP by tracheal perfusion for only once and simultaneously treated with an equal volume of Se. The doses of Se and AgNP were determined based on previous studies that had successfully established the experimental model[15]. Body weight was recorded every 3 days. After 2 weeks, all of the rats were anesthetized with isoflurane and subsequently euthanized. Blood samples were collected through cardiac puncture for serum preparation, while the aorta was isolated, cleaned using a 0.9% NaCl solution, ultimately stored for subsequent histopathological and biochemical analyses, including the assessment of biomarkers related to oxidative stress and inflammation gene expression.
HUVECs culture
The American Laboratory of Sciences (Catalog 8000) provided the HUVECs, which were then cultured in a specific medium for endothelial cells (ScienCell #1001). The medium was added with 5% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% endothelial cell growth factors (ECGS). Once the culture reached a 70-80% confluence, the cells were utilized for subsequent investigations.
Cell viability assay
The evaluation of cell viability was carried out by utilizing the Cell Counting Kit-8 (CCK-8) (AbMole, China) according to the procedures outlined by the manufacturer. HUVECs were cultivated in 96-well plates until they reached the desired confluence. Subsequently, they subjected to various treatments were incubated at 37°C for a duration of 24 h. Then each well was added with 10 μL of CCK-8 and allowed to incubate for additional 2 h. The absorbance was then determined employing 450 nm (ALLSHENG, Hangzhou, China).
Transmission electron microscopy (TEM) examination
The aortic tissues were fixed in a solution containing 2% glutaraldehyde for a period of 2 h. After this, they underwent three consecutive washes with 0.1M dimethyl sodium arsenate at intervals of 2 h. Afterwards, the tissues were post-fixed for 2 h in a solution containing 4% osmic acid, and then rinsed twice with 0.1M dimethyl sodium arsenate. To aid in the dehydration process, a series of escalating alcohol concentrations was applied to the tissues. It should be noted that all the forementioned procedures were conducted at a temperature of 4°C. Following the completion of the dehydration process, the samples were permeated with propylene oxide and then embedded in an epoxy resin. The subsequent step involved polymerizing the resin at 60°C for 48 h. To achieve ultra-thin sections, a diamond knife was used for sectioning purposes. These sections were subsequently stained using uranyl acetate and lead citrate. After the preparation process, we observed these sections and captured corresponding images using a TEM.
Histopathological examination
First, the samples were fixed in 4% paraformaldehyde (PFA) at 4℃ for 24 h, then dehydrated in different concentrations of ethanol solutions (100%, 95%, 90%, 80% and 70%) and embedded in paraffin wax after xylene transparency. Second, sections were then cut at 4 µm using a rotary slicer. Sections were examined microscopically by hematoxylin and eosin (H&E) staining and Masson’s trichrome staining.
Determination of oxidation indexes
After the treatment, blood specimens extracted from diverse groups were used to centrifugate at 3,000 g for a duration of 10 min for the isolation of plasma. The levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) in plasma were quantified using specialized biochemical reagent kits obtained from a commercial company (NanjingJiancheng, China).
ROS measurement in vivo and in vitro
ROS levels was detected by DCFH-DA according to test kits (Beyotime, China). For the in vitro experiments, HUVECs were initially cultured in 24-well plates with 3×104 cells/well. The cells with a confluence of 70–80% were treated with Se and AgNP for of 24 h. Subsequently, the cells were washed and incubated for 30 min at 37℃ in the presence of 10 μM DCFH-DA, ensuring darkness during the incubation. For the in vivo investigations, it is essential to first dewax the tissue samples. Subsequently, the dewaxed tissue should be rinsed 3 consecutive times with phosphate buffered saline (PBS), each rinse lasting for a duration of 5 min. To proceed further, 100 μL of a 10 μM solution of DCFH-DA should be supplemented onto each aorta tissue. Following this, the solution should be incubated at a temperature of 37℃ in darkness for an additional 30 min. After the incubation period, the tissue slices need to be rinsed thrice with 1× PBS for 5 min each time. To complete the staining process, a fluorescent sealing agent containing 4’,6-diamidino-2’-phenylindole (DAPI) should be added, and a cover glass should be used to seal the samples. Finally, the sections should be observed using an Olympus fluorescence microscope, and the resulting images should be collected for further analysis.
Immunohistochemistry and immunofluorescence staining
The tissues were prepared with an approximate thickness of 4 µm, and the paraffin surrounding the aortic tissues was eliminated using xylene. Then, the tissues underwent hydration using a range of ethanol concentrations (100%, 95%, 80%, and 70%) with each concentration lasting 5 min. Tissues were washed with PBS for 3 times at room temperature. Subsequently, the tissues were permeated with 0.5% Triton X-100 for 30 min, citrate antigen retrieval solution for 15 min, and finally blocked with goat-derived rabbit serum (Nakasugi Jinqiao, China) for 40 min. After the blocking step, the tissues were incubated overnight at 4°C with primary antibodies ICAM-1, VCAM-1, and NLRP3 separately for immunohistochemistry. Later, the tissues were treated with biotinylated secondary antibody at room temperature for 1 h. After washing with PBS 3 times, the tissue slices were exposed to diaminobenzidine (DAB) stock solution for 3 min and counterstained using hematoxylin for 2 min, resulting in blue-stained nuclei. For immunofluorescence, the sections were incubated overnight at 4°C with anti-ZO-1 (1:200; Affinity), anti-VCAM-1 (1:100; Abmart), anti-NLRP3 (1:100, Abmart), anti-Nrf2 (1:100, Affinity), and anti-CD31 (1:1000, Servicebio), respectively. After rinsing with PBS 3 times, the sections were separately incubated with goat anti-rabbit lgG /TRITC (1:200, ZSGB, China) or goat anti-mouse lgG /FITC (1:200, ZSGB, China). Following another round of washing with PBS 3 times, the sections were sealed with DAPI and captured using a fluorescence microscope (OLYMPUS, Japan). The software Image J was utilized to determine the percentage of the immune-stained areas.
Preparation of thoracic aortic rings
The isolated thoracic aorta was placed in a clean petri dish and injected with saline to remove the residual blood in the aorta. Then, the fat and connective tissue surrounding the aorta were then separated under a dissecting microscope. The isolated aorta was transected with straight scissors into a 4 mm long loop, and placed in 96-well plates with ABW® matrigel, as well as treated with AgNP and Se in a 37°C incubator. The fluid was changed in every 2 days. The number of budding rat aortic cyclic neovascularization was observed and counted on the day 4 under a high-power inverted microscope.
The in vivo measurement of vascular permeability
Evans blue is a highly water-soluble synthetic diazo dye with strong affinity for serum albumin and is a high molecular weight protein tracer in the blood. Under normal conditions, lower plasma albumin cannot penetrate the blood-brain barrier and is therefore often used to measure the integrity of the blood-brain barrier. We assessed vascular permeability in rat heart by slow injection 1% Evans blue solution (2 mg/kg) through tail vein of rats [20, 21]. Changes in the skin and mucosal membranes of the rats were observed, representative photographs were taken. After 2 h, all the rats were sacrificed simultaneously as soon as possible. Subsequently, saline was then injected to eliminate any remaining traces of blood along with the Evans blue solution within the cardiac region. The excised hearts were collected and placed in a 2.0 mL centrifuge tube with 1 mL of normal saline per 100 mg of tissue. The tissues were rapidly ground using a tissue homogenizer to make a homogenate, and then subjected to 1000 g centrifugation for 20 min in a 4°C ultracentrifuge. Following that, two portion of formamide were introduced to each tissue homogenate and subsequently immersed in a water bath maintained at 37°C for a duration of 24 h. This incubation period aimed to extract Evans blue from the tissues. The quantity of Evans blue present in the heart was determined using a UV-spectrophotometer, specifically measuring the absorbance at 620 nm.
Endothelial tube formation
An 80 μL volume of growth factor-reduced matrigel (ABW, China) was used to coat a 96-well cell culture plate, which was subsequently incubated at 37℃ for 30 min to facilitate matrigel polymerization. Following this, isolated primary HUVECs were seeded onto the wells coated with matrigel, and they were then cultured at 37℃ for 6 h in the presence of either AgNP (0.3 μg/mL) alone or both AgNP (0.3 μg/mL) and Se (6 µM) to observe tube formation. Tube formation was documented using an inverted microscope and analyzed utilizing the Image J software.
Endothelial cell migration
To evaluate the impact of AgNP on the movement and penetration of endothelial cells, transwell permeable supports from Corning incorporated (NY, USA) were employed. Initially, a serum-free medium containing 3 × 104 HUVECs was introduced into the upper chamber of the transwell system using 200 μL. Following that, the HUVECs were exposed to 0.3 μg/mL AgNP and either 0 μM or 6 μM Se for stimulation purposes. After a 24 h incubation period, the cells were fixed with 4% paraformaldehyde for 30 min and subsequently stained with 0.1% crystal violet for 10 min. The chambers were placed there after rinsed with PBS, while any nonmigratory or invasive cells were gently wiped off from the upper side of the chamber using a cotton bud. The migratory cells were digitally captured under an inverted microscope (OLYMPUS, Japan) at five randomly selected fields. To count the cells, the Image J software was utilized.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
Real-Time quantitative PCR was utilized to analyze the mRNA levels of ICAM-1, VCAM-1, endothelial nitric oxide synthase (eNOs), IL-6, TNF-α, IL-10, IL-1β, Nrf2, HO-1, NLRP3, ASC, caspase-1 and IL-18 in accordance with the provided guidelines, the RNA extraction kit was employed to isolate total RNA from the rat aorta tissue (Corning, USA). A transcript uni cDNA was synthesized by a commercial PCR kit (TransGen, China). The 2−ΔΔCt method was employed to cascade the relative mRNA levels, with GAPDH serving as the internal reference. Table 1 exhibited the primer sequences implemented in the present study.
Table 1
Enzyme‑linked immunosorbent assay (ELISA)
In this study, we utilized rat enzyme-linked immunosorbent assay (ELISA) kits from Proteintech (USA) to determine the levels of IL-6, TNF-α, IL-1β, and IL-10 as inflammatory mediators in the serum samples. Moreover, a commercial human ELISA kit from Abmart (China) was used to measure the same inflammatory factors in the cell culture supernatants.
Western blot
Proteins were extracted from the thoracic aorta through homogenization using cold RIPA lysis buffer, and total proteins were quantified using a BCA kit (KeyGen, China). An equal amount of total protein was then subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the resulting proteins were transferred onto PVDF membranes via electro transfer at a constant current of 200 mA for 45 min in the tris glycine methanol buffer. Then, the membranes were obstructed using 5% non-fat milk for a duration of 2 h at room temperature and subsequently incubated at a temperature of 4°C in the presence of primary antibodies, rabbit or mouse lgG raised against NLRP3 (1:1000, Abmart), Caspase-1 (1:000, Abmart), ASC (1:1000, Abmart), TLR4 (1:1000, Abmart), NF-κB (1:1000, Abmart), HMGB1 (1:1000, Abmart), TNF-α (1:1000, Servicebio), IL-1β (1:1000, Servicebio), IL-18 (1:1000, Abmart) and β-actin (1:5000, Servicebio) overnight. After that, the membrane underwent 3 successive rinses for a duration of 10 min each with buffered saline containing tween (TBST). Subsequently, the membrane was incubated for 1 h with secondary HRP-labeled goat anti-mouse/rabbit lgG (1:5000, ZSGB, China). Ultimately, after washing, the membrane was exposed to ECL substrate for detection (NCM, China). Utilizing ImageJ software, the western blots were subjected to analyses concerning band size and density. Throughout all experiments, β-actin served as the internal reference.
Statistical analysis
Statistical analysis was performed using Prism 8.01 software (GraphPad Software Inc., United States). To assess the statistical difference between experimental groups, a Two-way analysis of variance (ANOVA) was conducted, followed by the Turkey multiple-comparison test. P < 0.05 was statistically significant.