Silicon dioxide

Silicon dioxide (size distribution: 80% between 1 and 5 μm, 99% between 0.5 and 10 μm) was obtained from Sigma Co. (St. Louis, MO, USA). The particles were crushed for 3 h and then suspended in saline at a final concentration of 50 mg/mL. The silicon dioxide suspensions were autoclaved and mixed well before use.

Compositions determination of Fermented cordyceps powder

Fermented cordyceps powder (FCP) provided by Jiangxi Sino Pharmaceutical Co. LTD. (Nanchang, China), is a fermentation product of Paecilomyces hepialid (Cs-4) purified from Cordyceps sinensis (approval number: National medicine approval Z10890021; Factory batch number: 19090438). FCP is a light brown powder with a special aroma of lactones and a slightly bitter taste.

According to the quality standard of FCP in the 2020 edition of the Chinese Pharmacopoeia, the compositions of adenosine, guanosine, uridine and ergosterol were determined by high performance liquid chromatography (HPLC) (Agilent 1260 Infinity II). The standard curve method was chosen for quantification and the peak area and concentration were proportional. The dilutions of the mixed standard of adenosine, guanosine and uridine were 25, 12.5, 6.25, 0.25 and 0.125 µg/mL. The dilutions of ergosterol standard were 40, 20, 8.0, 4.0, and 0.8 µg/mL. References of adenosine (YZ-110879), guanosine (SG9310), uridine (SU8080) and ergosterol (YZ-111845) were purchased from Solarbio (Beijing, China).

For preparation of test samples for the determination of adenosine, guanosine and uridine, 5 mg of FCP was accurately weighed and 1 mL of 70% methanol was added. After weighing, the mixture was sonicated (power 250 W, frequency 40 kHz) for 20 min, removed to cool, and then reweighed with 70% methanol. The mixture was shaken, filtered. A total of 500 μL was taken to dry and dissolved by adding 500 μL of water. The samples was tested with HPLC and chromatograms were obtained.

For preparation of the test sample for the determination of ergosterol, 2 mg of FCP was accurately weighed and 1 mL of methanol was added. The mixture was weighed, sonicated (power 500 W, frequency 40 kHz) for 60 min, cooled, and reweighed with methanol. The mixture was filtered for testing, and chromatograms were recorded.

Chromatographic condition: Welch Ultimate column (PLUS C18) 250 × 4.6 mm, 5 μm, the temperature was 25 ℃, the flow rate 1 mL/min, and the sample size was 10 μL. The detection wavelength of adenosine, guanosine and uridine was 260 nm. Mobile phase A was 0.02 M potassium dihydrogen phosphate aqueous solution and mobile phase B was methanol. The detection wavelength of ergosterol was 283 nm. Mobile phase A was water and mobile phase B was methanol.

Rats and treatment

Healthy male Wistar rats (6–8 weeks old) were obtained from Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China) and raised in a specific pathogen-free (SPF) animal room (temperature 24 ± 2 ℃, humidity 40–70%). Food and water were taken at will. All animal treatment protocols and procedures were approved by the ethical committee of Shandong Academy of Occupational Health and Occupational Medicine (ethical batch number: SDZFY-EC-A-2020-09). Animal experiments were conducted according to Weatherall (2006) Report and NC3Rs Guidelines.

All animals were divided randomly into 4 groups (n = 30/group) including the saline-instilled group (saline), the silica-exposed group (silica), silica-exposed rats that intervened with FCP (300 mg/kg) (silica+ FCP 300) and silica-exposed rats that intervened with FCP (600 mg/kg) (silica+ FCP 600). After 3 days of adaptive feeding, the one-time non-exposure intratracheal instillation method was used for animal modeling. Rats in the saline-instilled group were given 1 mL of sterile saline and rats in the other three groups were given 1 mL of silicon dioxide suspension (50 mg/mL). After 24 h, rats in the two intervention groups received the corresponding dose of FCP daily by intragastric gavage, and rats in the saline-instilled group and the silica-exposed group received sterile saline instead of FCP. On day 7, 28 and 56 after treatment, 10 rats in each group were anesthetized by intraperitoneal injection of pentobarbital sodium and sacrificed. Lung tissues and peripheral blood (PB) were collected for further analysis.

Histological analysis

Lung tissues were fixed with 4% paraformaldehyde, and embedded in paraffin, then cut into 5 μm sections. Sections were dyed with hematoxylin and eosin (HE) to determine the level of inflammation and dyed with Masson staining solution to observe the expression of collagen fiber.

Analysis of the Th1, Th2, Th17 and Treg cells by Fluorescence-activated cell sorting (FACS)

Lymphocytes in PB were purified with lymphocyte separation medium (TBD science, Tianjin, China). Then the cells surface were stained with CD3-PerCP/Cy5.5 (Biolegend, 201418, USA), CD4-FITC (Invitrogen, 11-0040-82, USA) and CD25-APC (Biolegend, 202114, USA), followed by intracellular staining with IFN-γ-PE (Biolegend, 507806, USA), IL-4-PE (Biolegend, 511906, USA), IL-17A-PE (Invitrogen, 12–7177-81, USA) and Foxp3-PE (Invitrogen, 12-5773-82, USA) using the Transcription Factor Staining Buffer Set (BD Pharmingen, USA) and protocol. The FACS method was performed with the BD FACSCantoTM IIFlow Cytometer (BD Bioscience, USA). The Th1 subsets were recognized as CD3+CD4+ and IFN-γ+ cells, the Th2 subsets were recognized as CD3+CD4+ and IL-4+ cells. The Th17 subsets were recognized as CD3+CD4+ and IL-17A+ cells, while the CD4+ and CD25+ Foxp3+ strategy was used to recognize Treg subsets.

Quantitative real-time PCR (qRT-PCR) of BALF cells

BALF was obtained by tracheal injection of sterile saline into the lungs and aspirated back, 2 mL each time for six consecutive times. After centrifuged at 1000 g for 10 min at 4 ℃, the precipitation of BALF cells was washed twice in PBS. The total messenger RNA (mRNA) of BALF cells was extracted using the SPARKeasy Cell RNA Rapid Extraction Kit (SPARKjade, AC0205, China). With the SPARKscript II RT Plus Kit (SPARKjade, AG0304, China), the mRNA was reversely transcribed into cDNA. Then qRT-PCR was performed with the 2 × SYBR Green qPCR mix (SPARKjade, AH0104, China) and the Roche LightCycler 480 real-time quantitative PCR instrument (Roch, CH). The primers used are shown in Table1. The 2–ΔΔCT method was used for quantitative analysis with β-actin as a reference gene.

Table1 Primer sequences of RT-PCRImmunohistochemical analysis

After deparaffinization and rehydration, tissuesections were treated with sodium citrate buffer (0.01 M) in a pressure cooker for 2 min to retrieve antigens. Then the sections were soaked in 3% hydrogen peroxide for 10 min to remove endogenous peroxidase. Sections were incubated with sheep serum at room temperature for 1 h to block nonspecific binding and then incubated at 4 ℃ overnight with the following primary antibodies: Rabbit anti-T-bet antibody (Proteintech, 13,700-1-AP, 1:400, USA); Mouse anti-Gata3 antibody (abcam, ab282110, 1:2000, UK); Rabbit anti-RORγt antibody (Proteintech, 13,205-1-AP, 1: 200, USA); Rabbit anti-Foxp3 antibody (Novus, NB100-39002, 1:400, USA). Following incubation with horseradish peroxidase (HRP) conjugated secondary antibody (DD13: ivision™ Poly-HRP sheep anti-mouse/rabbit secondary antibody, TALENT, China) at room temperature for 30 min, the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution was used to detect signals and hematoxylin was used to redye the nucleus. The images were captured and then analyzed by Image J.

Western blot (WB) analysis

Total protein was extracted from lung tissue using RIPA lysis buffer (SPARKjade, EA0002, China). The concentration of the protein samples was detected with the BCA protein assay kit (SPARKjade, EC0001, China). The proteins were then separated by sodium dodecyl sulfate–polyacrylamide gels and transferred to a polyvinylidene fluoride membrane which was blocked with 5% BSA for 1.5 h at room temperature. Then the membrane was incubated overnight at 4 ℃ with the following specific primary antibodies: anti-β actin (proteintech, 20,536-1-AP, 1:2000, USA); anti-α smooth muscle actin (α-SMA) (cst, 14968 s, 1:2000, USA); antifibronectin (FN) (abcam, Ab45688, 1:5000, UK). After washing with TBST, the membrane was incubated with the goat anti-rabbit IgG (H + L) HRP specific secondary antibody (SPARKjade, EF0002, 1:5000, China) for 1 h at room temperature. The Tanon Automatic Image Analysis System (Tanon 5200, China) was used to detect the protein signal and β-actin served as internal reference. The optical density of the protein bands was analyzed using Image J.

Analysis of cytokines by enzyme-linked immunoassay (ELISA)

Cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1) in lung homogenates were tested using commercial ELISA kits (Biokits, ER01-96, ER03-96, ER02-96, ER10-96, China) according to the kit instructions.

Statistical analysis

All data were presented as mean ± standard error of the mean (SEM). SPSS (20.0) software was used for statistical analysis. One-way analysis of variance (ANOVA) and the Student–Newman–Keuls (SNK) test were used to analyze differences between groups. P-value less than 0.05 was considered difference statistically.