Particle characterization
The SiNPs used in this study were obtained from Jiangsu XFNANO Materials Tech Co., Ltd (XFI03, particle size: 20 nm, 99.0% purity), dispersed in distilled water as stock, and sonicated before use (160 W, 20 kHz, 5 min; KQ5200DE). Particle morphology was observed using a transmission electron microscope (TEM; 80 kV, H7650, Hitachi, Japan). Particle size was measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the average particle size and particle size distribution of the synthesized SiNPs were obtained using Origin 2022 (OriginLab Corporation, USA). The hydrodynamic diameter and zeta potential of the SiNPs in distilled water were measured using dynamic light scattering (DLS) (Brookhaven Instruments Corporation, New York, USA).
Animals And Treatments
All animal studies were conducted according to the guidelines for the care and use of laboratory animals of the National Institute of Health (China) and supervised by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (ethics review number: AEEI-2020-171). Beijing Charles River Laboratory Animal Technology Co., Ltd. (Beijing, China) provided 32 female Wistar rats that met specific pathogen-free standards, with an average weight of 200 ± 20 g. Before the experiment, the rats were adapted to the animal facility for 1–2 weeks, and their overall health status was monitored daily. Rats were then randomly divided into four groups (n = nine per group) and treated with either normal saline (control) or SiNPs (3.0, 6.0, and 12.0 mg/kg body weight) by intratracheal instillation (once every day for five days). The control group received an equivalent volume of normal saline. Dose selection for SiNPs exposure was based on a previous study. [24] According to the National Institute for Occupational Safety and Health standards, the daily inhalation of SiNPs in adult rats is 3.156 mg/kg. At the end of the experiment, the rats were fasted overnight, and blood, lung, and lymphangion samples were collected.
Determination Of Pulmonary Permeability
To evaluate pulmonary permeability, Evans blue (EB, Cat#: E6135, Macklin) was injected intravenously through the tail of the rats (2% EB solution in saline, 4 mL/kg) as previously described with a slight modification. [25] After 2 h of circulation, the rats were transcardially perfused with cold phosphate-buffered saline (0.01 M, PBS, pH 7.4) under deep anesthesia. Subsequently, the right lung tissue was separated and weighed, and formamide (Cat#: F809511, Macklin) was added at 2 mL/g tissue weight. After incubation at 37 ℃ for 24 h, the concentration of EB was measured using a spectrophotometer (Thermo Fisher Scientific) at 620 nm. Based on the standard curve of EB, the EB concentration in the lung tissue (µg/g) was calculated as follows:
EB concentration (µg/g) = EB concentration (µg/mL) × formamide capacity (mL) / lung tissue weight (g).
Histopathological Examination
Left lung tissues were fixed in a 4% paraformaldehyde solution and embedded in paraffin. Paraffin sections (5 µm thick) were sliced and fixed on glass slides. Tissue slices were subsequently subjected to hematoxylin-eosin staining for histopathological lesion observation. Finally, the slides were scanned using a Panoramic Digital Slide Scanner (Pannoramic, 3D HISTECH, Hungary). All sections were reviewed and analyzed by a pathologist in a blinded manner.
Immunohistological Analysis And Immunofluorescence Staining
Immunohistochemical analysis was performed to assess the expression of CD45 in the lungs. The paraffin sections were dewaxed, rehydrated, and endogenous peroxidase was blocked with 3% H2O2, then immersed in ethylenediaminetetraacetic acid buffer (pH = 9.0) for antigen retrieval via microwave heating. Sections were incubated overnight with the primary antibody for CD45 (1:2,000 dilution, Cat#: 20103-1-AP, Proteintech) diluted in 0.01 M PBS at 4 ℃. After incubation with the corresponding secondary antibody, the sections were stained with 3,3-diaminobenzidine as a chromogen and hematoxylin for nuclear counterstaining.
Immunofluorescence analysis was performed to assess the middle left lung lymph vessel density (LVD). Slices were immersed in antigen retrieval at a temperature greater than 95 ℃ for 10 min in a microwave, then cooled naturally. After blocking with 5% BSA, the tissues were incubated with primary antibodies against lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (1:200 dilution, Cat#: AF7939, R&D Systems) and CD31 (1:1,000 dilution, Cat#: GB11063-2, Servicebio) overnight at 4 ℃. Following three washes in PBST (0.1% Tween-20 in PBS), the slices were incubated with the secondary antibodies Alexa Fluor® 488-conjugated donkey anti-sheep (1:300 dilution, Cat#: ab150177, Abcam) and CY3-Tyramide (1:300 dilution, Cat#: G1223, Servicebio), and the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Cat#: G1012, Servicebio).
The slices were imaged using an automatic slice-scanning system (Pannoramic, 3Dhistech, Hungary). Ten images were randomly selected, and the integrated optical density (IOD) values of CD45 were measured using the Image-Pro Plus 6.0 (Media Cybernetics, MD, USA) software. Moreover, 20 images were randomly selected to calculate the lymph vessel density values of the lung using ImageJ.
Transmission Electron Microscopy Analysis
Ultrastructural alterations in the lymphangion tissues were observed using transmission electron microscopy (TEM; H7650, Hitachi, Japan). In short, the lymphangion tissue was fixed in 2.5% glutaraldehyde at 4°C overnight and post-fixed in osmic acid for 2 h. After dehydration with a gradient of ethanol and acetone, the samples were embedded in an epoxy resin. Finally, ultrathin sections were stained with 2% uranyl acetate and lead citrate and subjected to TEM for ultrastructural observation.
Western Blotting
Lung and lymphangion tissues were lysed in RIPA lysis buffer (Cat#: KGP9100, KeyGEN) containing PMSF (1 mM), a proteinase inhibitor, and a phosphatase inhibitor. After centrifugation at 15,000 × g for 15 min, the supernatant was collected and its concentration was determined using the BCA Protein Assay kit (Cat#: P0012, Beyotime) according to the manufacturer’s instructions. Proteins (20 µg per lane) were loaded and electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gels (Cat#: P0012A, Beyotime). The separated proteins were transferred electrophoretically onto polyvinylidene fluoride membranes (Immobilon-P, Millipore, MA, USA). The blots comprised 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (v/v) for 1 h at 25 ℃ and incubated overnight at 4 ℃ with the respective primary antibody: β-Tubulin (1:1,000; Cat#: TA-10, ZSGB-BIO), VEGFR3 (1:1,000; Cat#: A13304, ABclonal), VEGFC (1:1,000; Cat#: bs-1586R, Bioss), VEGFD (1:1,000; Cat#: ab155288, Abcam), Prospero-related homeobox 1 (PROX-1) (1:1,000; Cat#: ab199359, Abcam), LYVE-1 (1:1,000; Cat#: A4352, ABclonal), p-ERK (1:1,000; Cat#: 28733-1-AP, Proteintech), and ERK (1:1,000; Cat#: 51068-1-AP, Proteintech). The membranes were washed and incubated with the relevant secondary antibody [horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin G (1:8,000; Proteintech)]. Membranes were then visualized using an ultrasensitive ECL chemiluminescence kit (BeyoECL Moon, Cat#: P0018FM; Beyotime). Densitometric analysis of the blots was performed using the ImageJ software. The expression level of each protein was determined over at least three independent experiments.
Statistical analysis
Data were analyzed using GraphPad Prism 8.3.0 and expressed as the mean ± SD or mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) from at least three independent experiments. One-way analysis of variance for multiple comparisons was performed after confirming the normal distribution of the data. Nonparametric statistics were used when the above assumption was violated, followed by the Kruskal–Wallis test for multiple comparisons and Dunn’s post hoc test. Data from the two groups were analyzed using the Student's t-test. P < 0.05 was considered to be statistically significant.