To examine alterations in gene transcription during M.tb infection, we constructed an in vitro bacterial infection model (27). A specific antibody was employed to perform immunofluorescence staining of Ag85B, a conserved M.tb-specific protein (28). The findings revealed a distinct Ag85B positive signal in THP-1 cells ( Figure 1A ), indicating the ability of macrophages to phagocytose M.tb. High-throughput sequencing was conducted to identify the effector molecules regulated by M.tb. A total of 30971 genes were changed and visualized in a scatter plot ( Figure 1B ). The gene expression was visualized through heatmap analysis (a total of 1811 DE-genes were identified, including 934 upregulated and 877 downregulated genes) ( Figure 1C ). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed and the top 30 were displayed ( Figure 1D ). Gene Set Enrichment Analysis (GSEA) was performed to identify differentially regulated gene sets, the inflammatory and apoptosis signaling pathway were enriched ( Figure 1E ). As the TNF signaling pathway is the top enriched pathway and plays a crucial regulatory role in macrophages, we reasonably hypothesized that the TNF signaling pathway plays a vital role in M.tb-infected macrophages and selected it for further verification. The genes related to the TNF signaling pathway were visualized using a heat map ( Figure 1F ). The results above indicate that the TNF signaling pathway exhibited the most significant difference during M.tb infection.

TNF is a versatile cytokine with multifunctional roles in inflammation and cell death. TNFR1, as one of the receptors for TNF-α, is a death receptor and involved in the classical apoptosis (29). In order to investigate the inflammatory response to M.tb infection, serum levels of inflammatory factors were assessed by ELISA in patients with spinal tuberculosis, with healthy volunteers serving as the control group. The results revealed a significant upregulation of serum concentrations of IL-6 and TNF-α in patients with spinal tuberculosis compared to healthy controls ( Figure 2A ). Immunofluorescence staining of TUNEL and VDR was conducted, showing strong positive signals in patients with spinal tuberculosis compared to healthy controls ( Figure 2B ). These findings suggest that M.tb infection leads to increased expression of TNF-α, IL-6, and severe apoptosis.

To identify the effector molecules regulated by 1,25(OH)₂D₃, M.tb-infected cells were treated with a gradient concentration of 1,25(OH)₂D₃ for 24 hours. The cell supernatant was collected to detect the levels of TNF-α and cells were collected for high-throughput sequencing. The differential genes related to the TNF signaling pathway were visualized using a heat map ( Figure 3A ). Compared with the untreated group, different concentrations of 1,25(OH)₂D₃ could significantly restrain the secretion of TNF-α dose-dependently ( Figure 3B ). These results indicate that 1,25(OH)₂D₃ can suppress the M.tb-induced inflammatory response in a concentration-dependent manner. Meanwhile, qRT-PCR was performed to validate the sequencing results and identify specific genes associated with the TNF signaling pathway and apoptosis, as well as to assess the regulatory effect of 1,25(OH)₂D₃ in infected cells. Results indicated that M.tb infection markedly increased the mRNA expression of TNFR1, TRAF2, FADD, and TRADD, activating the TNF signaling pathway. Conversely, 1,25(OH)₂D₃ suppressed the aforementioned genes dose-dependently ( Figure 3C ). Additionally, M.tb infection significantly upregulated the mRNA expression of Caspase-3 and Caspase-8, thereby inducing apoptosis in macrophages. In contrast, 1,25(OH)₂D₃ exhibited an inhibitory effect on M.tb-induced apoptosis ( Figure 3D ). The above results provide preliminary evidence that 1,25(OH)₂D₃ inhibits Mtb-induced inflammation and apoptosis through the TNF signaling pathway in THP-1 cells.

To elucidate the effects of 1,25(OH)₂D₃ on the apoptosis of infected THP-1 cells, flow cytometry was applied for apoptosis detection in THP-1 cells 24h after infection. First, we performed the CCK-8 assay to determine the cytotoxic effects of varying concentrations of VD on PMA-treated THP-1 cells within 24 hours ( Supplementary Figure S1 ). The results revealed that Treatment with 1,25(OH)₂D₃ led to a significant inhibition of early apoptosis in infected cells compared to the control group (P<0.01). Specifically, 1,25(OH)₂D₃ appeared to reduce early apoptosis by approximately 3.01% at a concentration of 10^-6 M ( Figure 4A , Supplementary Figure S2 ). Western blot analysis indicated that 1,25(OH)₂D₃ notably increased VDR expression and suppressed apoptosis by downregulating Bax and upregulating Bcl2, ultimately inhibiting activated caspase-3. To explore the role of VDR, VDR siRNA was used to silence the expression of VDR in THP-1 cells, then pretreatment with 10^-6 mol/L concentration of 1,25(OH)₂D₃ for 24 hours, followed by M.tb infection for 4 hours. Results showed the effect mediated by 1,25(OH)₂D₃ was reversed upon VDR silencing ( Figure 4B ). To further explore the underlying mechanism, THP-1 cells were exposed to M.tb for 4 hours, followed by treatment with 1,25(OH)₂D₃ (10^-6 mol/L) for 0, 15, 30, and 60 minutes. Western blot results indicated that treatment with 1,25(OH)₂D₃ greatly reduced the ratio of p-IκB α/IκB α and p-p65/p65 ( Figure 4C ). Additionally, the immunofluorescence staining for p65 was carried out, demonstrating a marked increase in nuclear translocation of p65 upon M.tb treatment, whereas 1,25(OH)₂D₃ evidently suppressed its translocation. Notably, nuclear translocation of p65 was reinstated following VDR silencing ( Figure 4D ). These findings suggest that 1,25(OH)₂D₃ inhibited M.tb-induced apoptosis in THP-1 cells through the NF-κB signaling pathway.

M.tb infection of macrophages typically localizes to different parts of the body via the bloodstream, leading to the development of various extrapulmonary tuberculosis diseases. To validate the results, we further explored the effects of 1,25(OH)₂D₃ in vivo using a mouse model of M.tb infection through intravenous injection ( Figure 5A ) (30, 31). Due to the animal laboratory’s limited security level of P2+, we utilized BCG for the animal experiments. To mimic this process, we administered BCG and 1,25(OH)₂D₃ via tail vein injection. 24h post-injection, we obtained ocular blood samples from the mice to measure TNF-α levels using ELISA. The results demonstrated a substantial increase in TNF-α levels in the blood of mice following BCG infection (P<0.001). Additionally, treatment with 1,25(OH)₂D₃ markedly decreased the expression of TNF-α (P<0.01) ( Figure 5B ). In order to assess the expression of TNF-α in organs, tissue samples from the lungs, livers, spleens, and kidneys of all mouse groups were collected for RNA extraction and subsequent qRT-PCR analysis. The findings revealed a substantial and consistent elevation in TNF-α transcript levels in multiple organs following BCG infection across all groups of mice. Furthermore, treatment with 1,25(OH)₂D₃ led to a notable decrease in TNF-α transcript levels in the lungs, liver, and spleen, but not in the kidneys ( Figure 5C ). H&E staining results did not show a significant inflammatory response among these groups, potentially due to the short duration of the infection or the weak virulence of the BCG ( Supplementary Figure S1 ). To assess the effect of 1,25(OH)₂D₃ on apoptosis in vivo, immunofluorescence staining for TUNEL was conducted on liver, spleen and lung sections from all groups of mice. The results indicated that treatment with 1,25(OH)₂D₃ markedly attenuated apoptosis induced by BCG infection in various tissues ( Figure 5D ). These findings indicate that 1,25(OH)₂D₃ inhibited M.tb-induced inflammation and apoptosis in various organs in vivo. We demonstrated in vivo and in vitro that 1,25(OH)₂D₃ ameliorates the inflammatory milieu induced by M.tb infection. Next we explored the effect of 1,25(OH)₂D₃ on M.tb-induced osteolysis via a mouse model of joint tuberculosis. BCG and 1,25(OH)₂D₃ were injected into the joint cavity of mice via a microsyringe. H&E staining results showed that treatment of BCG resulted in massive inflammatory cell infiltration and 1,25(OH)₂D₃ effectively inhibited BCG-induced inflammation ( Figure 5E ). These findings illustrate that 1,25(OH)₂D₃ effectively ameliorates M.tb-induced inflammation and multiple organ damage in vivo.

The M. tuberculosis H37Rv standard strain (abbreviated as M.tb) was provided by Chongqing Public Health Medical Center, while the BCG strain was purchased from Shanghai Jingnuo. All strains were resuscitated on solid Middle Brook 7H11 medium (Becton Dickinson, USA, 283810) supplemented with 10% oleic-albumin-dextrose-catalase (OADC, Becton Dickinson, USA, 212351) and 0.5% glycerol (Solarbio, CHN, G8190). Individual colonies of M.tb and BCG were selected and cultured in Middlebrook 7H9 medium (Becton Dickinson, USA, 271310) supplemented with 10% OADC, 0.5% glycerol, and 0.05% Tween 80 (Sigma Aldrich, USA, P4780).

The THP-1 cell lines were purchased from ATCC and maintained in 1640 medium (Gibco, USA,12633020) supplemented with 10% fetal bovine serum (Gibco, USA, A5670701) and 1% Penicillin & Streptomycin solution (Solarbio,CHN,P1400) in a 5% CO₂ atmosphere at 37°C. The cells were seeded in 24-well plates at a density of 1 × 10⁵ cells/well and incubated with phorbol myristate (PMA, Sigma Aldrich, USA) at a concentration of 100 ng/ml overnight to ensure proper cell adherence. 1,25(OH)₂D₃ (Sigma-Aldrich, USA, d1530) with purity >95% was stored at −20°C in the light-protected condition. Adherent THP-1 cells were infected with M.tb at an MOI of 1 for 4 hours, and then washed three times with PBS to remove bacteria. Experiments involving live M.tb were performed in a P2+ facility.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, GER,74106). Paired-end libraries were synthesized using the TruSeq™ RNA Sample Preparation Kit (Illumina, USA, RS-122-2001) following the TruSeq™ RNA Sample Preparation Guide. In brief, poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Subsequently, the mRNA was fragmented into small pieces using divalent cations at 94°C for 8 min. The cleaved RNA fragments were then used to generate first-strand cDNA with reverse transcriptase and random primers. This was followed by the synthesis of second-strand cDNA using DNA Polymerase I and RNase H. The resulting cDNA fragments underwent an end repair process, the addition of a single ‘A’ base, and ligation of the adapters. The products were then purified and enriched with PCR to create the final cDNA library. The purified libraries were quantified using a Qubit^® 2.0 Fluorometer (Life Technologies, USA) and validated with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the molar concentration. Clusters were generated by cBot with the library diluted to 10 pM and then sequenced on the Illumina NovaSeq 6000 (Illumina, USA).

In order to analyze the secretion of inflammatory factors in serum and cell supernatant, ELISA kits for TNF-α (NeoBioscience, CHN, 3511-1H-6) and IL-6 (NeoBioscience, CHN, 3361-1A-6) were utilized. In brief, 100 μL of sample or standard was added to each well of a 96-well plate, and then incubated with anti-TNF-α or anti-IL-6 at 37°C for 90 min. Each well was incubated with streptavidin-HRP at 37°C for 30 min. After incubating with TMB and stop solution, the 96-well plate was measured at 450 nm using a microplate reader (Thermo Multiskan FC, GER). The absorbance was measured at 450 nm with correction for non-specific background at 570 nm in accordance with the manufacturer’s instructions.

Total RNA of tissues or cells was extracted using an RNA simple Total RNA Kit (ES science, CHN) and reverse‐transcribed (RT) with ReverTra Ace qPCR RT Master Mix (Takara, JAN). Quantitative reverse transcription polymerase chain reaction (PCR) was carried out using SYBR qPCR Mix (Takara, JAN). The reaction volume of the RT mixture was 20 μl, while the reaction volume of the PCR mixture was 20 μl (cDNA 1‐8 μl, MIX 10 μl, primer F 1 μl, primer R 1 μl, with DEPC H2O added to 20 μl). The experiments were conducted following the provided protocols. The sequences of the primers used are listed in Table 1 .

The pathological tissues from spinal tuberculosis intervertebral discs (study group) and the control tissues (negative controls from intervertebral disc herniation patients) were collected from patients undergoing curettage of spinal tuberculosis and non-tuberculosis patients with ethical approval(Approval ID:KY2024198) and according to the Army Medical University and Human Tissue Act ethical procedures. Then Immunofluorescence staining was performed for TUNEL (Beyotime, CHN, C1086) and VDR (CST, USA,12550S). The tissues were sliced into 2-mm sections and treated with 0.1% Triton X-100 for 5 min and then blocked with 10% goat normal serum for at least 30 min. Then dilute the primary antibodies with 1:200 in blocking buffer to incubate the sections for 1 h at 37°C. Goat Alexa Fluor 488 anti-Rabbit IgG (Bioss, CHN, bs-0295G-BF488), Alexa Fluor 647 Goat anti-Mouse IgG (Bioss, CHN, bs-0296G-BF647) (1:500), and DAPI (Solarbio, CHN, C0065) (1:1000) were then applied. The fluorescence signal was observed under a fluorescence microscope.

Apoptosis rate was detected using the Annexin V-FITC apoptosis detection kits (Biospes, CHN, BAD1001). Briefly, 1 × 10 ⁵/Test of infected THP-1 cells were resuspended in 100 μl binding buffer, then labeled with Annexin V-FITC (5 μl) and propidium iodide (PI) (5 μl) for 150min in the dark at room temperature. After 1 h, the green (Annexin V-FITC) and red (PI) fluorescence were examined by flow cytometry (NovoCyte, ACEA, USA). The examination wavelength was 488 nm, and the emission wavelength was 530 nm.

Briefly, equal amounts of total protein (30 µg) from different groups of samples were loaded for each lane and separated by 10% SDS-PAGE, followed by electroblotting by transferring protein to the PVDF membrane. The primary antibody target on Caspase-3, Caspase-8, VDR, Bax, Bcl-2 and GAPDH were used, and HRP-conjugate goat anti-rabbit IgG as a secondary antibody. The bands’ intensities were quantified using Image J after being conversed with the grayscale. Anti-GAPDH (AP0063) antibody was purchased from Biworld (Minnesota, USA), Anti-Caspase-3 (A19654) antibody was purchased from ABclonal (Wuhan, China), Anti-Caspase-8 (ab32125) antibody was purchased from Abcam (Cambridge, Britain), Anti-VDR (12550S) antibody was purchased from CST (Boston, USA), Anti-Bax (5023S) antibody was purchased from CST (Boston, USA), Anti-Bcl-2 (4223S) antibody was purchased from CST (Boston, USA). HRP-conjugated anti-rabbit secondary antibody (A0208) was purchased from Beyotime Biotechnology (Shanghai, China).

Male C57BL/6 mice (20–25 g) were purchased from the animal center of the Army Medical University. A total of 15 6-week-old C57/BL6 male mice were randomly distributed into three equal groups (n=5): vehicle (injection with PBS), BCG (injection with 1×10⁷ CFU/mouse) and BCG +1,25(OH)₂D₃ (injection with 1×10⁷ CFU/mouse BCG followed by 0.5 µg/g 1,25(OH)₂D₃). All compounds were administered via the tail vein and articular cavity. For cytokine level detection, serum and tissues were collected from mice 24h after 1,25(OH)₂D₃ treatment. To assess organ damage, tissues including lung, liver and spleen were collected 48h after 1,25(OH)₂D₃ treatment. Hematoxylin and eosin (H&E) staining was then performed according to the manufacturer’s instructions and the sections were observed using a light microscope.

All experimental data were analyzed using GraphPad Prism (version 9.0) and were shown as mean ± standard error of the mean (SEM). An unpaired Student’s t-test for two groups or one-way analysis of variance for multiple groups was used to analyze differences. The grey value of the Western blot protein bands was normalized with GAPDH or β‐actin. All experiments were independently repeated three times. Statistical significance was set at P<0.05.