A total of 16 male Balb/c mice (age, 6-8 weeks; weight, 20±5 g) were obtained from the Jackson Laboratory. All animal experiments and experimental protocols were approved by the Ethics Committee of Ningxia University (Yinchuan, China; approval no. 2020-013). The mice were maintained in a pathogen-free facility at a constant temperature (24±1˚C) and 50-55% humidity with a 12-h light/dark cycle and were allowed to eat and drink ad libitum. M.tb purchased from Chinese Center for Disease Control and Prevention was grown in Middlebrook 7H9 broth (BD Diagnostic; Becton, Dickinson and Company) supplemented with 10% OADC and 0.05% Tween-80 at 37˚C in a tissue incubator with an atmosphere of 5% CO₂. After one week of acclimatization, the mice were randomly divided into the following four treatment groups (n=4 mice/group): i) Control group (50 µl 0.9% normal saline administered intranasally); ii) M.tb group [50 µl M.tb (50 µg/ml) administered intranasally]; iii) vitamin D3 group [50 µl vitamin D3 (Beyotime Institute of Biotechnology; 5 µmol/ml) administered intranasally]; and iv) vitamin D3 + M.tb group [50 µl vitamin D3 (5 µmol/ml) and 50 µl M.tb (50 µg/ml) administered intranasally]. Intranasal administration was performed under 1.5-2% isoflurane anesthesia with 50 µl of the saline, M.tb and vitamin D3 (Beyotime, China) instilled into one nostril. Treatment was repeated once a day for seven days in each treatment group. The animals were placed into the induction box (1.5-2% isoflurane). Once the animals were completely anesthetized (3-5 min; animals did not attempt resume prone position), anesthesia was maintained using 0.5-1.0% isoflurane.

Mice were sacrificed by cervical dislocation at indicated time points (7 days after M.tb and vitamin D3 treatment). Subsequently, the lungs were extracted from the mice, washed once with PBS and fixed in 4% paraformaldehyde for 24 h at room temperature. the fixed tissue was embedded in paraffin and sliced into 4-µm-thick sections using a microtome. Samples were then stained with H&E for 10 min at room temperature and images captured with a light microscope (magnification, x100).

The monocytic THP-1 cell line (ATCC) was cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) in a 5% CO₂ incubator at 37˚C. Before incubation, the cells were seeded into a 6-well plate at a cell density of 2x10⁶ cell/well and treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) at 37˚C for 24 h to transform into adherent macrophages. A vision-based automatic cell counter was used to determine the total number of cells.

The MTT assay (Thermo Fisher Scientific, Inc.) was used to assess cell viability. THP-1 cells were seeded into a 96-well plate at a density of 5x10³ cells/well and treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) at 37˚C for 24 h to transform into adherent macrophages. Subsequently, the resulting THP-1 cells was treated with different concentrations of vitamin D3 (0, 5, 20, 50 and 100 µmol/ml) at 37˚C for 24 h or treated with vitamin D3 (5 µmol/ml) at 37˚C for 0, 6, 12, 24 and 36 h. Following incubation, 20 µl MTT solution was added to each well, according to the manufacturer's instructions. The plate was then transferred to an incubator for another 4 h at 37˚C; DMSO solution (100 µl) was subsequently added to each well to dissolve the purple formazan. Finally, the absorbance (560 nm) was measured using a microplate reader (Bio-Rad Laboratories, Inc.).

THP-1 cells were seeded at a density of 5x10³ cells/well in 96-well plates and treated with 100 ng/ml PMA at 37˚C for 24 h to transform into adherent macrophages. Then the cells of different treatment groups were incubated with M.tb (MOI:35) and vitamin D3 (5, 10 and 20 µmol/ml) at 37˚C for 24 h. Cytosolic Ca²⁺ concentration was quantified using Fluo-4 acetoxymethyl (AM; Thermo Fisher Scientific, Inc.). In brief, Fluo-4 AM was diluted in PBS and the cells were incubated at a final concentration of 1 µM for 30 min at 37˚C to ensure that Fluo-4 AM was fully converted into Fluo-4 intracellularly. Cells were then washed three times with PBS solution, and the fluorescence intensity was assessed by acquiring emissions at 494 nm using flow cytometry. (Thermo Fisher Scientific, Inc.). FlowJo version 10 software (FlowJo, LLC) was used for flow analysis. The result was compared with the control group.

M-PER Mammalian Protein extraction reagent (Thermo Fisher Scientific, Inc.) was used to extract total protein from cells in the different treatment groups. A BCA kit was used to determine protein concentration. Total protein (10 µg per lane) was mixed with 6X loading buffer and separated using SDS-PAGE on a 10% gel; separated proteins were subsequently transferred to a PVDF membrane. After blocking with Superblock Blocking Buffer (Thermo Fisher Scientific, Inc.) at 37˚C for 1 h, the membrane was incubated with the following primary antibodies: anti-LC3 (cat. no. 14600-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-sequestosome-1 (p62, cat. no. 18420-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-Beclin-1 (cat. no. 11306-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-autophagy-related 5 (ATG-5, cat. no. 66744-1-Ig; 1:1,000; ProteinTech Group, Inc.) and anti-AKT (cat. no. 60203-2-Ig; 1:1,000; ProteinTech Group, Inc.) overnight at 4˚C. The PVDF membranes were washed five times with TBS containing 0.02% Tween-20 (1,000 ml TBS and 2 ml Tween-20). Following the primary incubation membranes were incubated with secondary antibodies [HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L), cat. no. SA00001-1; 1:10,000; ProteinTech Group, Inc. and HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L), cat. no. SA00001-2; 1:10,000; ProteinTech Group, Inc.] at room temperature for 1 h. ECL chemiluminescence kit (Thermo Fisher Scientific, Inc.) was used to visualize protein bands. β-actin was used as an internal control. The intensity of protein bands was analyzed using ImageJ (version 146; National Institutes of Health).

Total RNA was extracted using the RNA Extraction Kit (Takara Biotechnology Co., Ltd.), and cDNA was synthesized using a High-Capacity cDNA Archive kit (Takara Biotechnology Co., Ltd.). The following temperature protocol was used for reverse transcription: 37˚C for 15 min and 85˚C for 5 sec. Subsequently, the acquired cDNA was subjected to qPCR using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) and a SYBR-Green PCR Kit (Takara Biotechnology Co. Ltd.). The following thermocycling conditions were used for qPCR: 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec and 65˚C for 30 sec. The primers were synthesized by the Shanghai Sango Biotechnology Co., Ltd., and primer sequences are presented in Table I. Relative expression levels was analyzed using the 2^-∆∆Cq method and normalized to β-actin as an internal control (15).

THP-1 cells were seeded at a density of 1x10⁵ cells/well into 6-well plates and treated with 100 ng/ml PMA at 37˚C for 24 h to transform into adherent macrophages. Then the cells of different treatment groups were incubated with M.tb (MOI:35) and vitamin D3 (5 µmol/ml) at 37˚C for 24 h. After being processed according to different treatment groups, the cells were washed three times at 37˚C with PBS or Hanks' balanced salt solution to remove the culture medium. Subsequently, 1 ml Fluo-4 AM (1 µM) fluorescent probe (Thermo Fisher Scientific, Inc.) was added to the cells and incubated for 40 min in the dark at 37˚C. The NucBlue^™ Fixed Cell ReadyProbes^™ Reagent (DAPI; 5 µM; Invitrogen; Thermo Fisher Scientific) was used to stain the nucleus. Finally, a confocal laser scanning microscope was used to detect the fluorescence of Fluo-4 to verify changes in intracellular Ca²⁺ concentration.

All experiments were repeated at least three times. All data were analyzed using the SPSS 13.0 software (SPSS, Inc.). Unpaired Student's t-tests were used to compare differences between two groups, whereas one-way ANOVA followed by Tukey's post hoc test was used to compare differences between more than two groups. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of vitamin D3 on THP-1 cell viability, the THP-1 cells were either treated with different concentrations of vitamin D3 (0, 5, 20, 50 or 100 µmol/ml) for 24 h (Fig. 1A) or treated with vitamin D3 (5 µmol/ml) for 0, 6, 12, 24 and 36 h (Fig. 1B). The results demonstrated that the viability of THP-1 cells was significantly decreased by vitamin D3 in a dose- and time-dependent manner compared with the respective control group; as vitamin D3 was dissolved in DMSO, cells treated with a culture medium of 0.03% DMSO were used as the control. It was demonstrated that when the vitamin D3 concentration reached 5 µmol/ml, cellular viability was statistically different from the control group; therefore, this concentration was chosen for subsequent experiments.

Impaired Ca²⁺ homeostasis can lead to cellular dysfunction and autophagy (16). Whether Ca²⁺ homeostasis of THP-1 cells infected by M.tb is altered under vitamin D3 treatment, and whether vitamin D3 could induce autophagy by inhibiting Ca²⁺ concentration was therefore investigated. To examine the Ca²⁺ homeostasis of THP-1 cells infected with M.tb following vitamin D3 treatment, the intracellular Ca²⁺ concentration was quantified using Fluo-4 AM-treated THP-1 cells. Compared with the control group, M.tb infection for 24 h induced an ~26% increase in Ca²⁺ concentration (Fig. 2A). Compared with the M.tb group, it was demonstrated that the vitamin D3 + M.tb (5, 10 and 20 µmol/ml) groups had a significant inhibitory effect on Ca²⁺ concentration. Immunofluorescence assay results also demonstrated that vitamin D3 treatment markedly attenuated Ca²⁺ concentration compared with the control and M.tb groups (Fig. 2B). Compared with the vitamin D3 group, the Ca²⁺ concentration in the vitamin D3 + M.tb group was markedly enhanced. These results suggested that vitamin D3 treatment inhibited Ca²⁺ concentration in THP-1 cells infected by the M.tb.

Vitamin D3 exhibited a significant inhibitory effect on the cellular viability of THP-1, aforementioned. However, whether vitamin D3 induced autophagy in THP-1 had not been explored. To determine whether vitamin D3 regulated autophagy in THP-1 cells, the protein expression levels of autophagy-associated proteins, p62, LC3, Beclin-1, ATG-5 and AKT, were analyzed by western blotting (Fig. 3). Compared to the M.tb group, treatment with vitamin D3 significantly increased the protein expression levels of p62, LC3Ⅱ/LC3Ⅰ, Beclin-1 and ATG-5 under M.tb infection (Fig. 3B-E). However, treatment with vitamin D3 significantly suppressed the protein expression levels of AKT under M.tb infection compared with the control (Fig. 3F). Moreover, the increase in LC3 and AMP activated protein kinase (AMPK) mRNA expression levels in the vitamin D3 and vitamin D3 + M.tb groups was higher compared with the M.tb group (Fig. 3G and H). Western blotting and RT-qPCR analyses indicated that vitamin D3 may stimulate autophagy in THP-1 cells. Taken together, these results suggested that vitamin D3 may inhibit M.tb infection by increasing autophagy in THP-1 cells.

After the Balb/c mice were treated intranasally with vitamin D3 and/or M.tb, pathological sections were produced and H&E staining was performed. Pulmonary tissue sections from the control group (Fig. 4A) exhibited clear alveolar structure without edema, congestion or inflammatory cell infiltrates in the interstitial fluid. However, pulmonary tissues from the M.tb group (Fig. 4B) were damaged and inflammatory cell infiltrates and pathological changes were present. In the vitamin D3 (Fig. 4C) and vitamin D3 + M.tb groups (Fig. 4D), nasal administration was repeated for 7 days. Compared with the M.tb group, the degree of alveolar wall damage and infiltrating inflammatory cells in the vitamin D3 + M.tb group markedly decreased. These results suggested that vitamin D3 could reduce lung damage following M.tb infection by promoting cell autophagy.