Chemicals and kits
Bleomycin was obtained from Chemex Company (Argentina), bovine serum albumin, Ellman’s reagent (DTNB), thiobarbituric acid and Bradford reagent were purchased from Sigma–Aldrich. Ammonium molybdate, butylated hydroxytoluene, trichloroacetic acid, buffered formalin, HCl and perchloric acid were purchased from Merck Company. Commercial glutathione peroxidase (GPX) and superoxide dismutase (SOD) kits were purchased from RANSEL, Randox Com,UK. TNF-α commercial enzyme-linked immunosorbentassay (ELISA) kit was provided by Hangzhou, Eastbiopharm.
BLM-induced lung fibrosis in experimental animals [23]
We purchased thirty-two male Sprague Dawley rats, weighing between 150 and 250 grammes, from Sanzyme Ltd. in GaghanPahad, Hyderabad. The rats were kept in conventional settings, with 12 hour cycles of light and dark, 24±1º C temperature and 65±10% humidity, inside cages made of polypropylene. Water from the tap and standard pelletized feed were given. The Institutional Animal Ethics Committee (Reg no: MRCP/CPCSEA/IAEC/2018-19/MPCOL/9) authorised the protocols for every pharmaceutical trial. The rats were divided into four groups equally and at random, and they received the following care:
Group 1: The animals received normal saline (0.5mL/d, p.o.) for 14 days.
Group 2: Syringic acid group; 50 mg/kg was continued intraperitoneally (i.p.) for 14 days,
Group 3: BLM was applied intratracheally (7.5 units/kg, single dose) on day 1. Normal saline (0.5 mL/d, p.o.) was given for 14 consecutive days.
Group 4: BLM was applied intratracheally (7.5 units/kg, single dose) on day 1. Syringic acid (50 mg/kg/d, i.p.) was given for 14 consecutive days.
Under ether anaesthesia, a single intratracheal injection of BLM was administered. Saline was given orally to the Control group in place of BLM. Rats were weighed before and after the trial began. Rat blood samples were obtained for haematological parameters following the tests. After receiving BLM injections for 14 days, every rat was killed. Following the animals' overdose on anesthesia-induced death, the lung tissue samples were cleaned in cold saline and weighed in order to determine the lung/body mass index. By dividing the lung weight (g) by the body weight (g) and multiplying the result by 100, the lung index was calculated. Thereafter employed for histological and biochemical examination. The right section of the lung was placed in liquid nitrogen and stored at −70 °C until the assay for thiobarbituric acid-reactive substances (TBARS) a lipid peroxidation product, superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT), and glutathione peroxidase (GPx) contents. The left part of the lung was placed in formaldehyde solution for routine histopathological examination by light microscopy.
Determination of hydroxyproline in the lung tissue
A colorimetric assay was used to determine the left lung's total collagen content [25, 26]. The left lung was dried at 80◦C until it achieved a steady weight. The dry lung was hydrolyzed overnight in a glass vial with 12-N HCl at 120C while being vacuum-operated. The sample volume was adjusted to 30 mL using distilled water, and the pH was adjusted to 7 using NaOH. After combining the sample solution (1.0 mL) with 1.0 mL of chloramine T solution (0.05 mol/L), the mixture was incubated for 20 minutes at room temperature. After adding a 20% dimethyl benzaldehyde solution to the mixture, it was incubated for 20 minutes at 60 °C. Every sample was analysed by measuring its absorbance at 557 nm. The results are expressed in micrograms of hydroxyproline per gram of wet lung weight using the standard hydroxyproline curve [24, 25].
Biochemical assays
Two hundred milligrams of frozen lung tissue specimen was dissected into pieces on dry ice, homogenized in 1.15 % KCl buffer (1:9, w/v) using a manual glass homogenizer for approximately 5min and flushed with centrifugation for approximately 10s to remove large debris. The supernatant was used for analysis.
Using a commercial kit, the activity of SOD was measured (RANSOD kit, Randox Com, UK). For SOD, absorption was measured at 412 nm and reported as (IU/mg protein). Goth's colorimetric approach was used to measure the catalase (CAT) activity. After homogenised lung tissue was incubated with H2O2, the reaction was stopped after 10 minutes by mixing in ammonium molybdate. While the ammonium molybdate reacts with the sample's residual H2O2 to form a yellow complex, the CAT enzyme breaks down the H2O2 in the samples to produce H2O and O2. Using a spectrophotometer, absorption was measured at 410 nm. The expression for CAT activity was (μmol H2O2/min/mg protein) [26]. A commercial kit (RANSEL kit, Randox Com, UK) based on the Paglia and Valentine method [27] was used to measure the GPX activity. At 340 nm, absorption was measured, and (IU/mg of protein) was the expression for enzyme activity. Using Ellman's approach, the GSH level in lung tissue was determined. Ellman's reagent was applied to 40μl of lung tissue homogenate after it had been combined with 2ml of buffer phosphate. The GSH content of the yellow complex was measured using a spectrophotometer set to 410 nm, and was reported as (nmol/mg protein) [28]. Thiobarbituric acid reactive substances (TBARS) production was used to measure the amount of lipid peroxidation [29]. To summarise, 0.8 ml of a solution containing 0.25N HCl, 0.375% (w/v) thiobarbituric acid, and 15% (w/v) trichloroacetic acid (TCA) was mixed with 0.2 ml of homogenised tissue. Centrifugation was used for five minutes at 5000 RPM to precipitate and remove the protein. After being transferred to test tubes containing 0.02% butylated hydroxyl toluene, the supernatants were bain-marie heated for 15 minutes at 100°C. After that, samples were chilled and centrifuged for five minutes at 2000 RPM to get rid of the precipitant. When measuring absorption at 532 nm, MDA concentrations were reported as (nmol/mgprotein).
Determination of nitric oxide (NO)
The Griess method was used to determine the nitrite levels, which serve as an indicator of NO generation. First, acetonitrile (1:2, v/v) was used to deproteinize tissue samples. Subsequently, 100 μL of supernatant and 100 μL of Griess reagent were applied to a microplate well. After that, samples were incubated in microplate wells for 30 minutes at 37 °C, and the absorbance of the samples was eventually found at 546 nm. Based on a typical linear curve established by 0–150 mol/ml sodium nitrite, the quantity of nitric oxide metabolites in the samples was ascertained. For tissue samples, the data were given in moles per gram (gr) and per litre (L) [30, 31].
Bronchoalveolar lavage Fluid differential cell count
The process of obtaining bronchoalveolar lavage fluid involved injecting 3 ml of saline three times, for a total of 9 ml, and then gently aspirating the fluid out of the lung after inserting an intratracheal catheter into a trachea. The recovery ratio of lavage fluid with this catheter was roughly 80% and did not show any discernible variation across the groups. Using a hemocytometer, the total number of cells in the bronchoalveolar lavage fluid was determined. Smear slides were produced and stained with Giemsa solution to allow for differential counts of leukocytes in the broncholaveolar lavage fluid. Three hundred cells were counted differently for each sample.
Measurement of tumor necrosis factor-α
Tumor necrosis factor-α concentration was measured using an enzyme-linked immunosorbent assay kit (Xpressbio life scientific products). The determinations were done according to the test kit instructions.
Histopathological studies
Inflammatory and fibrotic evidences in histopathology studies
The tissue from the left lung was blocked in paraffin and put in 10% buffered formalin for histopathological analysis. Hematoxylin and eosin (H&E) and Masson's trichrome (MT) were used to stain 4 μm sections in order to assess collagen deposition, alveolar thickness, and inflammatory cell infiltration. The Ashcroft score [33] measured fibrotic lesions from 0 to 8, while the Szapiel score [32] graded alveolitis and inflammation from 0 to 3. Grading of inflammatory and fibrotic lesions was done on three slides per rat, with ten fields each slide.
Statistical analysis
The Data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc.). The result was expressed as Mean ± SEM and analyzed using one-way ANOVA followed by Tukey–Kramer multiple comparison tests. Previously, the normal distribution of data was evaluated. Differences were considered statistically significant at (p < 0.05).