Table 1.
Description of research groups.
Fig 1.
Relationship between lung tissue concentration of CRAMP and dose of synthetic cathelicidin use for inhalation.
Protein concentration was investigated in homogenates of mouse lungs collected from untreated mice and from animals after a single exposure to synthetic cathelicidin at concentration 4, 6 or 8 times higher than physiological. Each single inhalation included 1 h exposure to PBS and 1 h exposure to appropriate concentration of synthetic cathelicidin. CRAMP concentration was determined using the ELISA method. Linear regression analysis of CRAMP concentration in response to synthetic cathelicidin inhalation was conducted. Studies were conducted in 4 research groups, each of which contained 4 animals. Each sample was analyzed in 4 replications. (control mice N = 4; treated mice per research group N = 4).
Fig 2.
Changes in immune cells composition in response to cathelicidin (CRAMP) and/or Saline Extract of Pantoea Agglomerans (SE-PA) treatment.
Cells number was investigated in lungs collected from untreated mice and mice exposed to investigated compounds for 14, 28 or 42 days using flow cytometry. The data are presented as mean of cell number ± SEM. Each research group contained 8 mice: 6 treated and 2 untreated animals. Samples were collected from all animals and analyzed in 2 replications. (control mice N = 16; treated mice per research group N = 6). * p < 0.05 vs. untreated; # p < 0.05 SE-PA+CRAMP 14 d./28 d. vs. SE-PA 14 d./28 d. (comparison within corresponding time points); @ SE-PA 28 d. + untreated 14 d. vs. SE-PA 28 d.; $ p < 0.05 SE-PA 28 d. + CRAMP 14 d. vs. SE-PA 28 d. + untreated 14 d.; one-way ANOVA test; Tuckey post-hoc test.
Fig 3.
Changes in cytokines expression induced by cathelicidin (CRAMP) and/or Saline Extract of Pantoea Agglomerans (SE-PA) treatment.
Protein concentration was investigated in homogenates of lungs collected from untreated mice and animals exposed to investigated compounds for 14, 28 or 42 days using the ELISA method. The data are presented as a mean of protein concentration ± SEM. Each research group contained 8 mice: 6 treated and 2 untreated animals. Samples were collected from all animals and analyzed in 2 replications. (control mice N = 20; treated mice per research group N = 6). * p < 0.05 vs. untreated; # p < 0.05 SE-PA+CRAMP 14 d./28 d. vs. SE-PA 14 d./28 d. (comparison within corresponding time points); @ SE-PA 28 d. + untreated 14 d. vs. SE-PA 28 d.; $ p < 0.05 SE-PA 28 d. + CRAMP 14 d. vs. SE-PA 28 d. + untreated 14 d.; one-way ANOVA test; Tuckey post-hoc test.
Fig 4.
Changes in the morphology of lung tissue of mice exposed to cathelicidin (CRAMP) and/or Saline Extract of Pantoea Agglomerans (SE-PA).
Lungs collected from untreated mice and animals exposed to investigated compounds for 14, 28 or 42 days were stained with haematoxylin and eosin (H&E), as well as Masson trichrome dyes (TRI), and examined under light microscopy at magnification 200×. Each research group contained 8 mice: 6 treated and 2 untreated animals. Samples were collected from all animals and analyzed. (control mice N = 16; treated mice per research group N = 6). A) Representative photographs of mouse lung sections stained with H&E. B) Representative photographs of mouse lung sections stained with TRI. C) Quantification of inflammation and fibrosis in collected mouse lung tissue after H&E and TRI staining. The histological scores were graded with 4 point scales: 0 = regular tissue, 1 = mild changes, 2 = moderate changes, 3 = significant changes. The data for histologic scores for 2 independent investigations are given as mean ± SEM of investigated items. * p < 0.05 vs. untreated; # p < 0.05 SE-PA+CRAMP 14 d./28 d. vs. SE-PA 14 d./28 d. (comparison within corresponding time points); $ p < 0.05 SE-PA 28 d. + CRAMP 14 d. vs. SE-PA 28 d. + untreated 14 d.; one-way ANOVA test; Tuckey post-hoc test.
Fig 5.
Alterations in collagen deposition in response to cathelicidin (CRAMP) and/or Saline Extract of Pantoea Agglomerans (SE-PA) treatment.
Protein concentration was investigated in homogenates of lungs collected from untreated mice and animals exposed to investigated compounds for 14, 28 or 42 days using the ELISA method. The data are presented as a mean of protein concentration ± SEM. Each research group contained 8 mice: 6 treated and 2 untreated animals. Samples were collected from all animals and analyzed in 2 replications. (control mice N = 20; treated mice per research group N = 6). * p < 0.05 vs. untreated; # p < 0.05 SE-PA+CRAMP 14 d./28 d. vs. SE-PA 14 d./28 d. (comparison within corresponding time points); @ SE-PA 28 d. + untreated 14 d. vs. SE-PA 28 d.; $ p < 0.05 SE-PA 28 d. + CRAMP 14 d. vs. SE-PA 28 d. + untreated 14 d.; one-way ANOVA test; Tuckey post-hoc test.
Fig 6.
Changes in mouse pulmonary function in response to cathelicidin (CRAMP) and/or Saline Extract of Pantoea Agglomerans (SE-PA) treatment.
The frequency of breathing (f) and tidal volume (TV) was investigated before treatment and after every 7 days of mice exposure to investigated compounds. The data were collected every 2 s for 15 min. The data are presented as a mean of tested ventilatory parameters ± SEM. Each research group contained 8 mice: 6 treated and 2 untreated animals. A pulmonary function test was conducted in all animals. * p < 0.05 vs. untreated; ^ treated vs. untreated (comparison within corresponding time points); # p < 0.05 SE-PA+CRAMP vs. SE-PA (comparison within corresponding time points); @ p < 0.05 SE-PA 28 d. + CRAMP 14 d. vs. SE-PA 28 d. + untreated 14 d. (comparison within corresponding time points); $ p < 0.05 SE-PA 28 d. + CRAMP 14 d./ SE-PA 28 d. + untreated 14 d vs. SE-PA (day 28). One-way ANOVA test; Tuckey post-hoc test.