2.1 Materials
2.1.1 Cells and subjects
Neutrophils: 6~8 weeks old healthy SD rats were reared to 280-300 g (50 rats) and anesthetized. The blood sample was collected from the abdominal aorta to obtain differentiated neutrophils in the peripheral blood.
Subjects: ZnO-NPs, white powder in appearance, 99.5% purity, 30 nm average particle size, were prepared in physiological saline to a total concentration of 10 mg/ml, and then ultrasonicated for 5 min with an ultrasonic cleaner to disperse the nanoparticles.
2.1.2 Main reagents
The reagents are presented as follows: IL-8 ELISA Kit (Wuhan KeLu Biological Company), Zinc Oxide Nano (Nanjing Haitai Nanomaterials Co., Ltd.), Protease Inhibitor Cocktail (ROCHE, Switzerland), Kodak Medical X-ray Film (Beijing Zhongke Terry Company), Indicator Detection Kit (Wuhan KeLu Biological Company), Protein Marker (Thermo, USA), IL-6 ELISA Kit, TNF-α ELISA Kit, LC3B antibody, P62 antibody, PBS solution, saline, SDS-PAGE gel preparation. Thermo), IL-6 ELISA Kit, TNF-α ELISA Kit, LC3B antibody, P62 antibody, PBS solution, saline, SDS-PAGE gel preparation kit, RIPA total protein lysate, BCA protein concentration determination kit, ECL chemiluminescence assay kit, 100*PMSF, phosphorylated protease Inhibitor, 5*Protein Loading Buffer, Lichon Red Staining Solution, TBS (powder), PBS (powder), Electrophoresis Solution (powder), Electrophoresis Solution (powder), Transmembrane Solution (powder), Antibody Elution Solution, Development and Fixing Solution, Skim Milk Powder, as well as BCA Protein Concentration Determination Kit (ASPEN, South Africa).
2.1.3 Main instruments
The instruments are presented as follows: Enzyme marker (Diatek Company), benchtop centrifuge (Shanghai Anting Scientific Instrument Factory), ice machine (Changshu Xueke Electric Co., Ltd.), constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.), electrophoresis instrument (Beijing Liuyi Instrument Factory), transfer electrophoresis instrument tank (Beijing Liuyi Instrument Factory), vertical electrophoresis tank (Beijing Liuyi Instrument Factory), enzyme marker (Diatek Company), enzyme marker (Diatek Company) Decolorization shaker (Beijing Liuyi Instrument Factory), water bath (Jintan Jiangnan Instrument Factory), as well as scanner (Canon Corporation).
2.2 Methods
2.2.1 Rat feeding and isolation and culture of peripheral blood neutrophils
Healthy SD experimental rats aged 6-8 weeks were employed, and the required weight of the rats reached 280-300 g. 3-5 ml of peripheral blood was collected from the rats using anticoagulated blood collection tubes and then introduced to 1 ml of lymphocyte isolation solution (performed in 5 ml centrifuge tubes). Centrifugation was performed at 500 g for at least 30 min at ambient temperature. Neutrophils located in the lower band were collected after centrifugation. In two layers, the first layer was monocytes, and the second layer was neutrophils. RP1640 diluted with pure water to 50% RP1640 was employed for resuspension of neutrophils, thus recovering cellular activity. Intermediate interval (5-10 min); centrifugation at 400g for at least 10 min was performed to remove the supernatant of the solution. Afterward, cell separation was performed to obtain neutrophils, and neutrophils were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum under conventional conditions in an electric thermostat incubator at 37℃, 5% CO2, and 95% humidity under suitable environmental conditions.
2.2.2 Effect of ZnO-NPs on the level of cell inflammation
Different concentrations of ZnO-NPs (0, 5, 10, 15 and 20 mg/L) were adopted to act on neutrophils, and the expression levels of the relevant inflammatory factor IL-8 were examined using the enzyme-linked immunosorbent assay (ELISA) reagent method.
2.2.3 Effect of ZnO-NPs on the level of cellular autophagy
Different concentrations of ZnO-NPs (0, 5, 10, 15 and 20 mg/L) were employed to act on neutrophils, and the changes in the expression levels of autophagy-associated protein LC3B were examined through the Western Blot analysis. (Western blot analysis: an appropriate amount of lung tissue was taken, total protein was extracted, and the protein content was obtained using the Bradford method. The separated proteins were transferred to the nitrocellulose membranes (NC membranes) at 10 V constant pressure for 35 min. 5% skim milk powder was closed at ambient temperature for 2 h. The NC membranes were washed three times with Tris-buffer containing 0.05% Tween-20 (TBST). The primary antibody solution at a dilution of 1:100 was added. The mixture was incubated overnight at 4°C, and peroxidase (HRP)-labeled primary antibody (1:1000) was added after TBST washing. The secondary antibody (1:1000 dilution) was added, and the mixture was incubated for 2 h at ambient temperature. The membrane was washed, and the chemiluminescence (electrochemiluminescence, ECL) reagent was added for color development exposure. The relative optical density value (target gene strip optical density value/β-actin strip optical density value) was adopted to express the relative expression intensity of the target protein, with β-actin as an internal reference for the amount of the upper sample.
2.2.4 Effect of altering the level of cellular autophagy on cellular inflammatory response
The appropriate action concentration of ZnO-NPs was selected in accordance with the results of the above experiments. The cells were pretreated with the autophagy inhibitor trimethyladenine (3-MA) at a concentration of 5 mmol/L and the autophagy activator rapamycin (RAPA) at a concentration of 200 nmol/L, respectively. Subsequently, the cells were and then stimulated with ZnO-NPs to examine the expression levels of autophagy-associated proteins LC3B and P62 and inflammatory factors (IL-8, IL-6, and IL-1β).
2.2.5 Statistical analysis methods
Data results are primarily expressed as mean ± standard deviation (x±s), and spss25.0 statistical software was employed for data collation and analysis. Multiple data groups were analyzed using one-way ANOVA, and the comparison between two data was drawn using LSD with a test level of α=0.05.