Goat anti-rabbit horseradish peroxidase (HRP) (sc-2030; CA, USA), anti-TNF-α (sc-52746), anti-nitrotyrosine (sc-32757), anti-CD 45 (sc-1178), and 3,3′-diaminobenzidine (DAB) substrate (7304) were procured from Santa Cruz Biotechnology Inc. (TX, USA). Polyclonal anti-TGF-β1 (AV 37894), anti-iNOS (N7782), 2′,7′-dichlorofluorescein diacetate (DCF-DA), diaminofluorescein diacetate (DAF-2), dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA), chloramine-T, sodium acetate, isopropanol, p-dimethylaminobenzaldehyde, perchloric acid, trans-4-hydroxy-l-proline standard, and all other required chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA). Mayer’s hematoxylin and eosin (H&E) stain was purchased from Fisher Scientific (Pittsburgh, PA, USA).

Female C57BL/6 mice, an established model for studying radiation-induced pulmonary injuries (23), were selected for the current study. Mice (24 ± 2 g), aged 8–10 weeks, taken from inbred colony (INMAS, Delhi, India), were used as per the protocols approved by Institutional Animal Ethics Committee (INM/IAEC/16/21), INMAS. Animals, maintained at controlled light and temperature conditions, housed in polypropylene cages containing sterile paddy husk were fed with standard food pellet (Amrut Laboratory Animal Feed, Maharashtra, India) and water ad libitum. Anesthetized mice were exposed to thoracic irradiation (11 Gy) by 60Co gamma irradiator (Cobalt Teletherapy Bhabhatron-II, Mumbai, India), in specific holder designed to immobilize the animals. Throughout the study, radiation dose was maintained at 0.83 Gy/min. Dosimetry was carried out with Fricke’s chemical dosimetry method by institutional radiation safety officers.

Randomly selected healthy animals were divided in four groups (six mice per group): non-irradiated control, G-003M only treated, 11 Gy irradiated and G-003M + 11 Gy. Group G-003M + 11 Gy was administered with G-003M intramuscularly (5 mg/kg body weight of animal), −1 h to 11 Gy thoracic gamma radiation (TGR). Experiments were repeated twice.

The formulation used in the current study, coded as G-003M, is a combination of two bioactive molecules podophyllotoxin and rutin purchased from Sigma Aldrich (St. Louis, MO, USA) in their 98% purity. To prepare G-003M freshly, both the compounds were dissolved in DMSO, which was 10% of the total injectable volume (20 µl in 200 µl of total injectable). Their molecular weight and chemical structure have already been revealed in a publication by Dutta et al. (13).

A separate group of six mice was used for survival and symptomatic monitoring studies. The animals were monitored daily for body weight, change in hair color, and survival. Survival was reported as percentage of animals surviving till 16 weeks after irradiation. The ratio of lung weight to body weight was used as lung weight coefficient (lung weight/body weight × 1,000). Mice from each treatment group were monitored for their breathing rate by using SA II (Model 1030, Small Animal Instruments, Inc., NY, USA) as per the manufacturer’s instructions. Breathing rate of six mice per group was recorded at 8 and 16 weeks posttreatment in conscious mice in relatively calm position to avoid experimental error.

After tracheotomy, bronchoalveolar lavage was performed in sacrificed animals using 200 µl of warmed sterile phosphate-buffered saline (PBS). The solution was instilled five times into the trachea, and fluid was withdrawn gently (24). The aspirated fluids were pooled, immersed immediately in slurry of ice and centrifuged for 10 min at 2,000 rpm. The supernatants were separated into aliquots and kept frozen at −80°C until analysis. BALF cells were resuspended in 1 ml PBS and quantified with a hemocytometer by the conventional method. Out of the total BALF cells, number of macrophages was calculated based upon their morphological identification such as large size, eccentric nuclei, and abundant cytoplasm. BALF was centrifuged; smeared on clean glass slides, stained with May-Grünwald–Giemsa and observed in blind manner under the microscope to confirm the ratio of macrophages and leukocytes, counted in hemocytometer.

Total protein, LDH, and ALP activity was measured in serum/BALF samples of experimental animals. BALF was collected from sacrificed mice at different time intervals after treatment. For serum analysis, blood collected in plain vials was centrifugated at 5,000 rpm for 10 min at 4°C. Separated serum was stored at −20°C until analysis. The level of TP, LDH, and ALP was measured in all the experimental mice by using fully automatic Biochemistry Analyzer (Erba; Model No: EM-360).

The IL-6 and TNF-α contents were detected in BALF of differentially treated mice by flow based Kits BDTM Cytometric Bead array (CBA) Flex Set (BD Biosciences, USA) according to the manufacturer’s instructions.

Intracellular ROS/NO generation was measured in BALF cells by flow cytometry and fluorescence microscopy, 1 h posttreatment. ROS were detected using ROS-sensitive fluorescent dye DCF-DA (Sigma Co., St. Louis, MO, USA). The cells (2 × 10⁴/ml), washed with PBS, were incubated for 15 min with DCF-DA (10 µM) dye. Generation of ROS was studied using flow cytometer (BD Accuri™ C6, Becton Dickinson Biosciences, USA) and imaged with fluorescent microscope (Model No. BX-63, Olympus, Japan). To study NO formation, the cells were stained with NO-sensitive fluorescent dye 4,5-DAF-2. Data were analyzed with Image J software, and photographs were processed using Adobe Photoshop software (San Jose, CA, USA).

Proteins isolated from the frozen lung tissues were quantified by Bradford method (25). Denatured protein was subjected to 12% SDS-PAGE and transferred to Whatman PROTRAN nitrocellulose transfer membrane (Sigma). After blocking the non-specific sites with 5% (w/v) skimmed milk for 2 h, the membranes were incubated overnight at 4°C with primary antibodies anti-TGF-β1 (1:1,000)/iNOS (1:1,000)/nitrotyrosine (1:1,000). The membranes were then incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000) for 2 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system. Densitometry was performed on the resulting autoradiograph using Image Lab software of BioRad Gel Documentation System (Gel Doc XR; Cat. No. 1708195).

Whole lung was excised from animals dissected at different time intervals. Lung tissues, individually from all the mice, were weighed after blotting. Lung weight coefficient was calculated by using the formula (lung weight/body weight × 1,000). For histological and immunohistochemical studies, lung tissues were fixed in 10% formalin. In separate groups of animals, whole lung was used for determining Hpl content.

Differentially treated mice from all the groups were dissected at different time intervals following treatment. The lung tissues, excised individually from each mouse, were fixed in 10% formalin solution and dehydrated in series of alcohol. Paraffin-embedded tissues were sectioned at thickness of 5 µm with a semiautomatic microtome (Spencers, Gurgaon, India). The slides were stained with H&E to evaluate architectural changes in lung tissues. The extent of damage was assessed by Eldh et al. method (26). Images were obtained by using digital camera mounted on BX-63, Olympus microscope. Quantification of damage was performed by scoring from 0 to 4 based on the amount of area affected by interstitial inflammation, alveolar wall thickening, and peribronchial inflammation (26).

The sections stained with Masson’s trichrome were subjected to Ashcroft score for evaluating lung fibrosis (27). Severity of the fibrosis was graded and scored on a scale of 0–8. Ten fields per sections from the individual mice were randomly selected and scored at 400× magnification. Assessment was performed in double-blind fashion. After whole section examination, fibrosis score was evaluated as mean score of all the fields.

Hydroxyproline content in lung tissues of all the differentially treated mice was determined by colorimetric assay (28). Briefly, individual tissue was homogenized with 2 ml distilled water and incubated with 125 µl of 50% TCA on ice for 20 min. After centrifugation, the pellet was mixed with 1 ml hydrochloric acid (2 N) and baked at 110°C for 14–18 h until charred and dry. The acid dried samples were then re-suspended in 2 ml deionized water and mixed with 500 µl 1.4% chloramine-T in 0.5 M sodium acetate/10% isopropanol. After 20 min incubation at room temperature, 500 µl of Ehrlich’s solution (1.0 M p-dimethylaminobenzaldehyde in 70% isopropanol/30% perchloric acid) was added, mixed and incubated at 65°C for 15 min. Absorbance of each individual sample was measured at 550 nm. Concentration of lung Hpl was calculated by Hpl standard curve prepared by serial dilutions of trans-4-hydroxy-l-proline standard (Sigma, St. Louis, MO, USA). Samples were assayed in triplicate.

Cell death was detected by the TUNEL method using TUNEL apoptosis detection kit (catalog no. 17-141; Upstate Technology, Lake Placid, NY, USA). Briefly, 5 µm sections were deparaffinized, dehydrated through series of alcohol and incubated with proteinase K [1/24 (v/v) in PBS] for 15–30 min at 37°C. After stopping proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase buffer, biotin-dUTP, and terminal deoxynucleotidyl transferase in ratio of 90:5:5, respectively) for 60 min at 37°C. Following washing and blocking, avidin–fluorescein isothiocyanate (1:10) was applied to the samples and incubated in dark for 30 min at 37°C. The slides were washed with PBS, counterstained with propidium iodide, and visualized under fluorescent microscope (Model No. BX-63, Olympus, Japan). Dead cells were quantified by counting the TUNEL+ cells using pseudo-colored overlay images prepared by ImageJ software (National Institutes of Health, Bethesda, MD, USA). Percentage of TUNEL+ cells was obtained against the total number of nucleated cells (propidium iodide counter stained) in 10 different fields per tissue section.

Lung tissue sections were processed to evaluate immunohistochemical expression of CD45, TGF-β1, TNF-α, iNOS, and nitrotyrosine. Deparaffinized sections were passed through graded series of ethanol (100, 90, 70, and 50%), and slides were washed in distilled water. Subsequently, the sections were incubated with 0.3% H₂O₂ for 30 min to block endogenous peroxidase activity before performing heat-induced antigen retrieval. After antigen retrieval in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), slides were washed, and expression of antigens was detected by applying DAB substrate solution. The sections were counterstained with hematoxylin, dehydrated, mounted, and scored for immunopositive cells under a light microscope. Inflammatory infiltrates were evaluated by counting CD45+ aggregates in 10 randomly selected fields, per lung tissue section. Results were obtained by scoring the sections blindly by at least two individuals.

Statistical tool SPSS (Statistical Package for Social Science; Windows version 10.0) packed program was used for finding significance values of data. Values were expressed as mean values ± SEM and for analysis of differences between the groups. One-way ANOVA was used with Newman–Keuls post hoc test. Statistical differences are presented at probability levels of p < 0.05, <0.01, and <0.001.

Radiation-induced pneumonopathy is associated with body weight loss and compromised survival. Therefore, we evaluated the effect of G-003M on survival, body weight, and other symptomatic features of irradiated mice. Percent survival in thoracic-irradiated C57BL/6 mice was measured in all the experimental groups. In radiation only group, all the animals survived up to 8 weeks postirradiation; however, in 10 weeks survival reduced to 83.33%. Survival in this group further dropped to 66.50% by 14 weeks (Figure 1A). In G-003M pretreated groups, all the animals survived up to the last time point of the study (16 weeks of experimentation) (Figure 1A). The body weight of control and G-003M only treated mice increased in natural way with time. However, in TGR treated group, weight loss was insignificant in first 4 weeks of radiation exposure. A remarkable fall (18–20%) in body weight was recorded within 8–16 weeks in this group (Figure 1B). In G-003M pretreated group, weight loss was much less (8–10%; p < 0.001) in comparison to the corresponding radiation-treated group (Figure 1B). After 8 weeks of irradiation, hair of the C57BL/6 mice turned gray in the thoracic region, the area of exposure (Figure 1C). In irradiated group, this gray color band was extremely apparent in comparison to G-003M treated mice. G-003M only treatment exhibited same hair color as in control group.

Radiation manifests lung injury in the form of acute pneumonitis and fibrosis. Development of fibrosis in lungs usually results in difficult breathing and increase in breathing rate in experimental animals as well as in humans (29, 30). In this study, increased breathing rate, yet not significant, was observed in irradiated animals by 8 weeks of radiation exposure, and it was markedly higher (fourfold; p < 0.001) at 16 weeks in comparison to respective untreated controls of the same age (Figure 1D). However, in G-003M-administered mice, breathing rate was much less (p < 0.001; G-003M + 11 Gy vs. 11 Gy) when compared with irradiated mice at 16 weeks of experimentation (Figure 1D). Treatment of G-003M only exhibited normal breathing rate in mice at all the time intervals.

Radiation induces damage predominantly by generation of ROS and NO in cellular milieu. In the current study, formation of ROS and NO was measured in BALF cells of experimental mice at 1 h post-TGR exposure. As evident in Figures 2A–C, ROS formation was found to be markedly (p < 0.001) elevated in BALF cells of TGR-exposed mice in comparison to controls. NO production, measured by using fluorescent dye 4,5-DAF-2, was also prominently raised in irradiated group (Figures 2D,F). However, pre-irradiation administration of G-003M significantly (p < 0.001) countered both ROS and NO production in BALF cells of irradiated mice. In G-003M only treated group no ROS and NO formation was evident at 1 h after treatment.

Radiation-induced nitrosative stress was assessed by expression of iNOS and nitrotyrosine in lungs of experimental mice. Peroxynitrite (ONOO⁻), a RNS, is detected in the tissues as nitrotyrosine, which is a stable product formed by reaction of ONOO⁻ with tyrosine. Figures 3A,B show iNOS and nitrotyrosine expression measured by immunohistochemistry and immunoblotting techniques in different treatment groups. Immunohistochemical study revealed that both iNOS and nitrotyrosine staining was not evident in lungs of control mice but significantly apparent in irradiated animals (Figure 3A). iNOS expression increased from first week and maximum at 8 weeks of posttreatment. Nitrotyrosine staining was intense in alveolar macrophages and at the site of injury (Figure 3A). Immunoblotting of iNOS and nitrotyrosine in lung tissues further validate these findings. Intense band of iNOS and nitrotyrosine was detected in irradiated group in comparison to controls (Figures 3B–D). However, iNOS and nitrotyrosine expression was not as marked as that of irradiated groups with the pretreatment of G-003M (Figures 3A,B). Expression of iNOS and nitrotyrosine was similar to controls in animals that were treated with G-003M only.

Free radicals produced by radiation play an important causative role in cell death. In the current investigation, cell death was detected in lung tissues by using TUNEL assay. As evident in Figures 4A,B, remarkable increase in the number of dead (TUNEL+) pulmonary cells was observed in 11 Gy thoracic-irradiated group as compared to untreated controls. Administration of G-003M formulation, 1 h prior to radiation, indicated significant (p < 0.001) reduction in the number of TUNEL+ pulmonary cells showing efficacious function of formulation against radiation-induced cell death (Figures 4A,B). No TUNEL+ cells were observed in control and G-003M only treatment group.

Recruitment of inflammatory cells into the lung has been associated with radiation-induced pneumonitis. Anti-inflammatory effect of G-003M formulation was evaluated by measuring BALF cells in lungs of irradiated mice. Nearly sixfold increase was seen in BALF cellular counts at 1 week of radiation exposure (Figures 5A–C). The number of these inflammatory cells amplified further and was maximum (about 14-fold increase; p < 0.001, 11 Gy vs. controls) at 16 weeks of study in this group. When number of macrophages was calculated, marked (p < 0.001) increase was observed from 4 to 16 weeks in irradiated group as compared to their respective controls. Interestingly, G-003M-pretreated mice showed significantly less number of macrophages and other BALF cells at all the time intervals of study when compared with irradiated groups. However, values were still higher in comparison to respective controls (Figures 5B,C). The number of BALF cells was found similar to controls in G-003M only treatment group at all the time intervals.

Radiation induces endothelial injury resulting in increased vascular permeability in lungs. Lung permeability and cellular damage were assessed in the current study by evaluating TP content, LDH, and ALP activity in serum/BALF of experimental mice. TGR resulted markedly enhanced (p < 0.001) level of proteins in BALF of irradiated groups in the first week of treatment (Table 1). The level of protein continued to rise significantly in BALF of irradiated animals with the advancement of time. No change in protein level was recorded in serum samples of irradiated animals. TGR-exposed mice showed nearly 1.5-fold increase in LDH activity in serum and BALF samples even in first week of treatment (Table 1). The level of LDH was found to increase in this group up to 16 weeks of study in both serum and BALF samples. No significant increase in ALP activity was observed within 4 weeks in BALF of irradiated animals. However, the activity increased significantly (p < 0.001) at 8 weeks and found to be very high (twofold increase) at 16 weeks of study in this group. Pretreatment of G-003M significantly declined the level of TP, LDH, and ALP in BALF/serum of irradiated animals at all the time points of study. However, values of protein, LDH, and ALP in BALF of irradiated animals were higher in comparison to respective controls (Table 1). G-003M only treatment exhibited no significant changes in the level of TP, LDH, and ALP in BALF/serum of mice at any studied time interval.

Pulmonary inflammation and fibrosis were estimated by histological studies. H&E and Masson’s trichrome-stained slides were observed for radiation-induced inflammatory cells infiltration in pulmonary alveoli and appearance of fibrosis at later phase. Infiltration of inflammatory cells and alveolar wall thickening was observed in lungs of irradiated mice from 1 to 8 weeks postirradiation (Figure 6A, c,g). Congestion of the alveoli, presence of foamy macrophages around the bronchioles and severe interstitial edema was recorded in the lungs from 4 to 8 weeks of postirradiation (Figure 6A, k). Lung edema measured by calculating lung weight coefficient was found maximum at 8 weeks postirradiation treatment (Figure 6B). At later phase (8–16 weeks), acute pneumonitis was followed by marked fibrotic changes as evident in H&E stained sections (Figure 6, o). Lung damage score was maximum in irradiated animals at 16 weeks postexposure (Figure 6C). However, pre-administration of G-003M showed significantly less interstitial edema, infiltration of inflammatory cells, and alveolar wall thickening in irradiated lungs (Figure 6, d,h,l,p). The formulation significantly (p < 0.01) lowered the values of lung weight coefficient in irradiated mice at all the time intervals, indirectly indicating reduction in radiation-induced pneumonedema. Lung damage score was also significantly minimized by G-003M pretreatment (Figure 6C). However, lung architecture was not completely normalized in this group in the study period. No pathological alteration in lung architecture was observed in the animals of G-003M only group.

To confirm infiltration of inflammatory cells in lungs after radiation exposure, immunostaining for common leukocyte marker CD45 was applied. Radiation caused a prominent increase (p < 0.001; 11 Gy vs. controls) in CD45+ cell aggregates in lungs of mice by 4–8 weeks after exposure (Figures 6D,E). These cells were predominantly distributed around the vesicular vessels and also found scattered in alveolar spaces. Significantly (p < 0.05) low number of CD45+ cell aggregates in the lungs of G-003M-pretreated mice indicated less inflammation in comparison to irradiated (Figures 6D,E). CD45-staining intensity in G-003M only group was similar to controls at all the time intervals.

Lung fibrosis was also studied by histological grading of fibrosis according to the criteria of Ashcroft et al. (27), using Masson’s trichrome-staining method (Figures 7A,B). Extensive deposition of collagen, as evident by light blue staining of collagen fibers around vessels and bronchioles, was observed in lungs of irradiated mice (Figure 7A). However, G-003M significantly attenuated the deposition of collagen in lungs of irradiated mice indicating its anti-fibrotic potential (Figure 7A).

Hydroxyproline content, the major constituent of collagen, was also measured in the lungs of experimental mice. No significant difference was recorded in Hpl content up to 8 weeks after radiation treatment in comparison to controls (Figure 7C). However, Hpl content was found remarkably increased (about 10-fold) at 16 weeks of TGR exposure. G-003M pretreatment had significantly (p < 0.001) lowered Hpl content in lungs of irradiated animals (Figure 7C). As a whole, the results indicated anti-inflammatory and anti-fibrotic effects of G-003M formulation in lungs of irradiated mice. Hpl content was similar to controls in G-003M only treatment group at all the studied time intervals.

Transforming growth factor-β1 is the key cytokine involved in progression of fibrosis. In the current study, expression of TGF-β1 was observed in lungs of mice by immunohistochemistry and further confirmed by western blotting (Figures 8A,B). Immunohistochemical staining revealed stronger expression of this cytokine in alveolar macrophages and lymphocyte of irradiated lungs as compared to controls (Figure 8A). The intensity of expression was significantly higher at 8 weeks. Figure 8B demonstrates expression of TGF-β1 by western blotting in lungs of mice sacrificed at 8 weeks postirradiation. Significant increase in band intensity was observed in irradiated lungs in comparison to controls. Pretreatment of G-003M significantly declined the expression of this cytokine in 11 Gy-irradiated lung tissues (Figure 8). However, the expression of TGF-β1 in this group was still higher than controls. No significant alteration in the expression of TGF-β1 was seen in lungs of mice treated with G-003M only.

Tumor necrosis factor-α and IL-6 are the major pro-inflammatory cytokines involved in lung inflammation. The concentration of TNF-α and IL-6 was measured in BALF of experimental mice at 4 and 8 weeks posttreatment by using flow based Kits BDTM CBA Flex Set (BD Biosciences, USA). The level of TNF-α was very high (p < 0.001) at 4 weeks (22.78 ± 3.50 pg/ml) and increased (119.41 ± 10.50 pg/ml) further by 8 weeks in BALF of irradiated animals when compared to controls (2.00 ± 0.10 pg/ml) (Figure 8D). TNF-α expression was also located in lung tissues by immunohistochemistry (Figure 8C). Intense expression of this cytokine was observed in irradiated lungs at 8 weeks, confirming our previous finding. However, G-003M pretreatment significantly (p < 0.001) reduced (10.4 ± 2.10 pg/ml at 4 weeks and 20.58 ± 3.50 pg/ml at 8 weeks) the level of TNF-α in BALF as well as its immunohistochemical expression in lungs of irradiated animals (Figures 8C,D).

Interleukin-6 concentration was also increased remarkably in BALF of irradiated animals in comparison to controls (2.80 ± 0.50 pg/ml). The level of IL-6 raised at 4 weeks (22.60 ± 2.50 pg/ml) and then declined at 8 weeks (15.96 ± 2.80 pg/ml) in this group (Figure 8E). G-003M significantly (p < 0.001) reduced (11.76 ± 2.2 pg/ml at 4 weeks and 2.95 ± 2.22 pg/ml at 8 weeks) the level of this inflammatory cytokine in BALF of irradiated animals (Figure 8E). No significant change in TNF-α and IL-6 was observed by G-003M only treatment in comparison to controls.