Silicon dioxide (0.5–10 µm), curcumin (diferuloylmethane), DCFDA (dichlorofluorescindiacetate), vanadium chloride (VCl3), Gelatin, hydroxyproline and Glutaraldehyde Grade 1 used as an electron microscope fixative (Sigma-Aldrich ,St Louis, MO, USA). Phosphotungstic Acid, Phosphomolybdic Acid, Acid Fuchsin were purchased from Sisco Research Laboratories (Mumbai, India) and Aniline Blue was purchased from Fisher scientific (Hampton, New Hampshire, United States).
Mice (Swiss strain, 25-30gram) were obtained from Animal facility of Banaras Hindu University, Varanasi, India and accustomed for two weeks under standard conditions oflight and dark cycle and were on lab chow diet with water ad libitum.Institutional Animal Ethical Committee, Banaras Hindu University, Varanasi, India has approved all the procedures of animal maintenance.
2.3 Preparation of silica particles
Crystalline silica (cSiO2) particles was sterilised at 120°C overnight then sonicated for 15 min in 200 mg/ml in autoclaved saline solution. Prior to intranasal administration of silica suspension, it was sterilised. Equal volume of autoclaved sterile saline was administered in control mice.
2.4 Experimental design
All mice were arbitarily allocated into 5 groupswith 5 mice each: Control group (saline only), crystalline Silica-induced group (cSiO2 only), curcumin group (cSiO2 + Cur), vehicle (cSiO2 + DMSO) group and Dexa group (cSiO2+dexamethasone). Mice model of Silicosis was developed by intranasal administration of crystalline Silica (cSiO2) for different durations, mainly 7, 14 and 21 days (every alternate day) to mimic the silicosis in human. Each duration of silicosis (7, 14 and 21 days) was thoroughly investigated by evaluating lung histopathology and biochemical parameters and silicosis features were observed in mice of 21 days of silica exposure, therefore, this duration was finally selected for detailed study. Curcumin was dissolved in Dimethyl Sulphoxide (DMSO) and given 10µl to each mouse (10 mg/kg, i.n., 5 µl/ nostril) an hour prior to silica administration. Dexamethasone (5 mg/kg, i.p.) was used as standard drug.
2.5 BALF and lung sample collection
After 24 hours of last intranasal silica administration, blood was collected via retro orbital bleedingandserum was separated by centrifugation and stored (at -20°C)to study biochemical parameters. Lungs were washed thrice by using 1 ml of sterile phosphate-buffered saline (PBS) and bronchoalveolar fluid (BALF) was collected,washed (at 3000 rpm for 15 minutes at 4ºC) and supernatant was stored (-80°C) for further analysis. Cell pellet was used for total and differential cell count. The lung lobes were separated and washed in chilled phosphate buffer saline solution. Some part of the lung lobes were fixed with 10% Neutral buffer formalin and rest were stored (-80°C) for biochemical analysis.
2.6 Reactive oxygen determination in BALF cell suspension
Reactive oxygen species (ROS) was measured using method standardised previously . BALF was washed with PBS thrice and the cell pellet was collected by centrifugation. DCFDA (10 mM) was added to the BALF pellet with equal amount of cells, thereafter incubation in dark at 37ºC for half an hour. Microplate fluorescence reader was used to measure fluorescence at excitation (485 nm)and emission (530 nm)wavelengths. Fluorescence intensity in arbitrary units was presented as ROS level.
2.7 Nitrite level measurement
Nitrite/Nitrate level in serum was analysed using griess reagent with some modifications. BALF supernatant (100 µl) was mixed with VCl3(0.8 gm/ ml in 1 N HCl;100 µl), Then freshly prepared Griess reagent (100 µl of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1‐Naphthyl) ethylenediaminedihydrochloride in water) was added. The reaction mixture was incubated at 37ºC for 30 min and absorbance was read at 540 nm.
2.8 Lung histology for airway inflammation and collagen deposition
Lungs were removed, washed in cold sterile PBS and then two lobes were fixed by using 10% neutral buffer formalin. The left lobe was fixed in glutaraldehyde for electron microscopy. Remaining lobes were stored (in -80ºC) for further biochemical analysis.After embedding in paraffinwax,lung sections(5 µm) were cut using microtome, stained with H&E for inflammatory cell infiltration. To study collagen deposition and fibrosis, masson's trichome stained lung sections were analyzed.
2.9 Transmission electron microscopy(TEM)
Immediately after sacrifice,lung tissue pieces(1 mm3) were made and fixed in 2% glutaraldehyde at 4°C,washedin 0.1 M phosphate buffer, pH 7.4for transmission electron microscopy (TEM). The sample was then taken to AIRF, JawaharLal University, New Delhi, India for further analysis(TEM).
2.10 Malondialdehyde assay (MDA)
Malondialdehyde (MDA) level was measured in lung tissue homogenate by thiobarbituric acid active substances (TBARS) using previously described method.Lung tissue homogenate in potassium phosphate buffer (10 %; pH 7.4) was made and mixed with (8.1% SDS, 375 µl of 20 % acetic acid) and 8.1 % thiobarbituric acid. After boiling for 1 h, it wascooled at room temperature to get pink colour. Distilled water (250 µl) was added followed by pyridine and n-butanol (1.25 ml of 1:1 solution). Mixture was separated by centrifuging at 2000 rpm for 10min and two layers were obtained and the absorbance of upper layer was read at 532 nm, and MDA concentration was expressed (moles / milligram).
2.11 Eosinophil peroxidase (EPO) activity
Eosinophil peroxidase (EPO), an enzyme is stored in the granules of eosinophils and its activity was determined using previously described method . Equal concentrations ofBALF (100 µl in PBS) and substrate solution (consisting of 0.1 mM O-phenylene-diamine-dihidrochloride, 0.1% of Triton X-100, 1 mM hydrogen peroxide in 0.05 M Tris–HCl pH maintained at 8.0, wastaken and further incubated for 30 min at 37°C. Sulphuric acid (50 µl, 1 M) was added to stop this reaction. Absorbance wasread at 490 nm using ELISA plate reader.
2.12 Myeloperoxidase (MPO) activity
Myeloperoxidase (MPO) is an enzymestored in the granulesof neutrophils. Infiltration of these cells as inflammation marker was analysed by Myeloperoxidase (MPO) activitywhich was quantified by previously described method with minor changes. Lung homogenate was prepared in potassium phosphate buffer (50 mM;pH 6.0), containing 0.5 % cetyl trimethyl ammonium bromide (CTAB) centrifuged at 12,000 rpm for 30 min,frozen (-80°C) and thawed. This process of freezing and thawing was repeated (thrice). Supernatant (20 µl)was mixed with reaction mixture containing 0.167 mg/ml Odianisidine dihydrochloride (ODD) and 0.002 % hydrogen peroxide (H2O2) in 50 mM potassium phosphate buffer. Change in absorbance for every 20 minutes at 460 nm was noted to measure the MPO activity using micro plate reader and the unit was denoted as MPO units per milligram of tissue.
2.13 Hydroxyproline Determination in Lungs
Hydroxyproline, an amino acid is the precursor and the main component in collagen, a triple alpha helix. Collagen content was measured using hydroxyproline content measurement .Acid digestion with 12 N hydrogen chloride (HCl) of lung tissue collagen is a good biochemical index for collagen content measurement.A part of lungs were homogenised (10% w/v in phosphate buffer saline). Equal volume of lung homogenate sample was acid hydrolysed (12 N HCl at 120°C for 16–18 hours). Tissue homogenate supernatant (50 µl) was collected after centrifugation at 13000 rpm for 15 min at 4ºC, suspended in citrate-acetate buffer. Citrate buffer consists of glacial acetic acid (1.25%), sodium acetate (7.24%), and citric acid of 5 %, 7.24% and 3.4% of sodium hydroxide (NaOH) in distilled water (pH 6.0). Freshly prepared 1.4 % chloramine-T solution with 10% N-propanol was added to the sample and kept at room temperature for 20 min. After addition of freshly prepared Ehrlich’s solution (4-dimethylaminobenzaldehyde dissolved in 18.6 ml of n- propanol and 7.8 ml of 70% perchloric acid ), sample was heated (at 65°C for 15 min). Optical density was taken at 550 nm and concentration of Hydroxyproline present in total lung tissue was calculated in µg units.
2.14 Collagenase Activity determination using gelatin zymography
MMP-9 protease activity was measured using gelatin zymography in bronchoalveolar fluid (BALF). Gelatin (10 mg/ml) was dissolved in resolving gel (10% SDS-PAGE), BALF supernatant protein (50 µg) was loaded in wells and gel was run at 4°C. After washing the gel for thrice (10–15 minutes to remove the sodium dodecyl sulphate (SDS), in renaturing buffer containing 2.5% Triton X-100, gel was incubatedfor 48 h at 37°C and washed in incubation buffer containing 50 mM Tris–Hydrogen chloride, 50 mM Tris base, 5 mM Calcium chloride, 0.2 M sodium chloride and 0.02% NaN3 (pH 7.5). The gel was stained with Coomassie Brilliant Blue R 250 stain for 10–20 min and destained till clear white bands visible in the stained gel. ImageJ software was used to analyse bands via densitometry.
2.15 Statistical Analysis
ANOVA was used to measure significant changes between two or more than two groups’ using Tukey’s and post hoc test comparison. Level of significance was considered at p < 0.05 by using SPSS 16 software. The values are presented as the mean ± SEM.