## 4 Discussion

Table 2: Changes in murine pulmonary function in response to inhalations with vitamin D3 metabolites. The mice were chronically exposed to calcidiol at the concentration of 100 pg/g (Group 5) or calcitriol at the concentration of 5 pg/g (Group 6). Inhalations were performed daily for 14 (subgroup A) or 28 (subgroup B) consecutive days. As the control, untreated mice from Group 1 (main control) and Group 2 were used. Animals from Group 1 were given the diet with a standard cholecalciferol level (0.5 IU/g), while the mice from other research groups were given a diet with a reduced cholecalciferol level (0.05 IU/g). Respiratory parameters were investigated using the Non-invasive Airway Mechanics Plethysmograph. The examinations were conducted at the end of every single experiment. Data were collected every 2 s for 10 min. The data are presented as a mean of tested ventilatory parameters \(\pm\) SD. Statistically significant differences between research groups were determined using a one-way ANOVA with a Tukey post hoc test and marked in the table. * \(p\) < 0.05; ** \(p\) < 0.01 vs. main control (Group 1). a cholecalciferol content of 0.5 IU/g, while to induce vitamin D deficiency the mice were given feed with a 10-times lower amount of cholecalciferol. Performed studies revealed that the calcitriol concentration in 3-month-old male mice strain C57 BL/6J on the standard diet was 30.31 pg/mg in the lungs and 132.24 pg/mL in the serum. On the other hand, dietary cholecalciferol restriction significantly decreased the calcitriol amount in the investigated mice to the following levels: 18.20 pg/mg (lung tissue) and 98.61 pg/mL (serum). The presented serum results correspond with data collected by [PERSON] et al. from male and female murine strain FVB after 3 months of feeding with aliments containing 0.5 IU/g or 0.05 IU/g cholecalciferol, which revealed serum concentrations of 1,25(OH)\({}_{2}\)-vitamin D3, 111 \(\pm\) 16 pg/mL, and 78 \(\pm\) 3 pg/mL, respectively [17]. On the contrary, [PERSON] et al. examined the effect of diets containing cholecalciferol in amounts from 25 to 1000 IU/kg on calcitriol serum levels in 14-week male murine strain C57 BL/6, which showed that animals given 50 and 400 IU/kg vitamin D with diet had the following levels of 1,25(OH)\({}_{2}\)-vitamin D3 in serum: 60 pmol/l (an almost 4-times lower concentration than that obtained in our study) and 160 pmol/l (an almost 2-times lower concentration than that obtained in our study) [18]. Nevertheless, another investigation conducted by [PERSON] et al. in a 10-week female murine strain C57 BL/6, fed with a diet containing cholecalciferol in amounts from 400 IU/kg to 20,000 IU/kg, indicated that animals on the diet with the lowest investigated vitamin D content had calcitriol serum levels around 300 pmol/l, which corresponds to our results. It needs to be highlighted that [PERSON] et al. reported a negative correlation between vitamin D diet intake (cholecalciferol amount over 100 IU/kg in male mice and over 400 IU/kg in female mice) and calcitriol serum level [18]. Cited research [17; 18] confirmed the correctness of the vitamin D doses in animal feed selected by our team to create a physiological and pathological level of calcitriol in the murine body. The concentrations of vitamin D3 metabolites used in the presented study were inspired by research conducted by [PERSON] et al. [19], which, according to the best of our knowledge, is the one of three studies that investigated the influence of inhalation of calcidiol and calcitriol on the lungs. Despite the fact that [PERSON]'s research was conducted in a different animal model (neonatal rats with unknown vitamin D3 status) and the aim of the study differed from ours (improve lung maturation), because of the similar concept of the studies (animals chronic exposure to vitamin D3 metabolites administered by nebulization) and in the light of limited knowledge in this area, we decided to use vitamin D3 metabolites in a similar concentration range [19].