## 3 Results
Examination of calcitriol levels in murine lung tissue and serum indicated that one-week exposure to 25(OH)-vitamin D3 at a concentration of 100 pg/g or 1,25(OH)\({}_{2}\)-vitamin D3 at a concentration of 5 pg/g was able to restore the physiological level of calcitriol in vitamin-D3-deficient mice. In order to confirm the stability of the observed beneficial changes, the time of murine exposure to the analyzed metabolites of vitamin D3 was extended to 2 and 4 weeks. As presented in Figure 2 and Table S1, murine chronic exposures to investigated compounds in the above-mentioned doses effectively restored the endogenous level of calcitriol in lung tissue samples collected from mice with vitamin D deficiency. The calcitriol level recorded in mice on the diet with 0.05 IU/g cholecystiferol, exposed for 14 and 28 days to 100 pg/g 25(OH)-vitamin D3, elevated from 18.20 pg/mg to 30.29 and 31.83 pg/mg, while in response to 5 pg/g 1,25(OH)\({}_{2}\)-vitamin D3 they reached the following values: 31.08 and 32.09 pg/mg, respectively. Moreover, serum samples examination (Figure 2 and Table S1) revealed that calcidiol at the concentration of 100 pg/g administered via nebulization for 14 or 28 days to vitamin D3-deficient mice, increased the calcitriol concentrations to 129.18 and 132.12 pg/mL, respectively. Similarly, chronic exposure of mice to exogenous calcitriol at the concentration of 5 pg/g also restored the physiological serum level of this compound; recorded 1,25(OH)\({}_{2}\)-vitamin D3 concentrations after 14 and 28 days of treatment were 124.03 and 126.82 pg/mL. A pulmonary function test was conducted as the last step of the study. As presented in Table 2, none of the investigated respiratory parameters depended on the amount of vitamin D in the diet of the mice. Similarly, chronic exposure of the mice to vitamin D3 metabolites did not impact on TV, Ti, Te, frequency of breathing, inspiratory and expiratory flow rates, or duty cycles of inspiration and expiration. On the contrary, mice on the diet with reduced cholecystiferol, exposed for 14 days to calcidiol at the concentration of 100 pg/g, demonstrated a significant increase in MV to the level 109.27 mL/min (Group 5A) from the value of 82.94 mL/min recorded in untreated mice on the diet with standard vitamin D content (Group 1). Disorders in pulmonary function were also observed after comparison data collected from Group 1 and animals that inhaled calcitriol at the concentration of 5 pg/g for 14 days (Group 6A). In the mentioned case, an increase was observed in PIF, PEF, and EF50 from 4.726 mL/s, 3.900 mL/s, and 3.536 mL/s to 6.377 mL/s, 5.307 mL/s, and 4.621 mL/s, respectively. It needs to be highlighted that extending the exposure time of the mice to the tested compounds did not increase the reported differences but, on the contrary, restored the balance disturbed by the 2-week inhalation. Moreover, it is also worth stressing that comparing results from treated and untreated mice fed the same diet did not show any significant changes in the investigated respiratory parameters. Figure 2: Restoration of calcitriol physiological level in mice with induced vitamin D3 deficiency in response to inhalation with vitamin D3 metabolites. The mice were chronically exposed to calcidiol at a concentration of 100 pg/g or calcitriol at a concentration of 5 pg/g. Inhalations were performed daily for 14 or 28 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 cholecaliferol level (0.5 IU/g), while the mice from other research groups were given a diet with a reduced cholecaliferol level (0.05 IU/g). Calcitriol concentration was determined by using the ELISA method in both lung homogenates and serum samples collected from all animals. Data are presented as a mean of calcitriol concentration \(\pm\) SEM of at least 10 measurements. ***\(p<\) 0.001 vs. main control (Group 1); ***\(p<\) 0.001 vs. control (Group 2); Analyzed using a one-way ANOVA test and Tukey post hoc test.