The study participants were recruited from the Department of Orthopedics at Ningxia Medical University General Hospital and included 40 patients with confirmed STB who were diagnosed between January 2021 and June 2022. The patient group consisted of 25 males and 15 females, with an age range of 37 to 73 years (mean age: 53.5 ± 4.6 years). A control group of 40 healthy volunteers was also enrolled, comprising 20 males and 20 females, aged 40 to 72 years (mean age: 51.3 ± 5.5 years). No statistically significant differences in gender distribution or age were observed between the two groups (P > 0.05). Patients with STB were enrolled if they had a confirmed diagnosis supported by histopathology or a combination of typical imaging features, etiological evidence and clinical manifestations, were aged 35-75 years, had not received prior anti-tuberculosis treatment, and provided written informed consent. Patients with other spinal infections, concurrent active tuberculosis at other sites, autoimmune diseases, malignancies, severe organ dysfunction, recent immunosuppressant use, or pregnancy/lactation were excluded. Healthy controls were frequency-matched for age and gender, had no history of spinal diseases or infectious disorders, showed no abnormalities in routine examinations, and provided informed consent. Individuals with a history of tuberculosis, chronic inflammatory diseases, or recent immunomodulatory drug use were excluded from the control group.

For proteomic screening, three subjects were randomly selected from the 40 participants in each group (STB and control). Subsequent validation of candidate proteins via qRT-PCR and ELISA was performed using the full cohort of 40 subjects per group. The study protocol was reviewed and approved by the Research Ethics Committee of Ningxia Medical University General Hospital, which determined that the research conformed to established ethical standards (Approval No. KYLL-2021-932). Written informed consent was obtained from all participants prior to enrollment.

Proteomic profiling was performed on both clinical plasma samples and cultured THP-1 macrophages to identify differentially expressed proteins and their associated signaling pathways. For clinical samples, peripheral plasma was collected from 3 patients with STB and 3 age- and sex-matched healthy controls; whole blood was centrifuged at 3,000 rpm for 10 min at 4 °C, and the supernatant plasma was stored at -80 °C until use. For cellular experiments, THP-1 macrophages were allocated to four groups: blank control, BCG-stimulated, LBP-knockdown control (LBP knockdown without BCG treatment), and BCG-stimulated + LBP-knockdown groups, with total cellular proteins extracted using RIPA lysis buffer supplemented with a protease inhibitor cocktail. All protein samples were quantified via the bicinchoninic acid (BCA) assay, digested into peptides with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS); specifically, plasma protein samples were labeled with isobaric tags for relative and absolute quantification (iTRAQ) before detection, while cellular protein samples were processed via the filter-aided sample preparation (FASP) method for label-free quantification. Raw mass spectrometry data were searched against the human UniProt reference database for protein identification, and differentially expressed proteins between paired groups were screened with consistent thresholds of fold change > 1.20 or < 0.83 and P < 0.05.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted separately on two sets of differentially expressed proteins: those identified from clinical plasma proteomic profiling, and those derived from pairwise comparisons of different THP-1 macrophage treatment groups. The clusterProfiler R package was used for enrichment analysis to delineate the signaling pathways associated with protein expression alterations under each experimental condition. The significance threshold was set at P < 0.05, fully consistent with the differential protein screening criteria described in Section iTRAQ-based proteomic analysis.

Total RNA from blood samples was extracted using the TRIzol method (Invitrogen, USA), and cDNA was synthesized by reverse transcription of RNA using the PrimeScript RT reagent Kit (Thermo, USA). The obtained cDNA was used for qRT-PCR validation experiments on the LightCycler480II quantitative platform (Roche, USA), using β-actin as an internal reference. All 16 differentially expressed proteins identified by proteomic screening were validated at the mRNA level, and the corresponding primer sequences are listed in Table 1. The reaction system consisted of 1 µL of template cDNA, 0.4 µL each of the upstream and downstream primers, 8.2 µL of ddH2O, and 10 µL of SYBR MIX, with a total volume of 20 µL. PCR conditions were as follows: 95 °C for 3 min; 95 °C for 5 s; 60 °C for 30 s, for a total of 40 cycles. The obtained data were analyzed using the 2-ΔΔCt method to calculate the relative gene expression levels.

Peripheral whole blood (3-5 mL) was collected from each subject, and centrifuged at 3,000 rpm for 10 min at 4 °C within 1 h after collection. The plasma supernatant was harvested and stored at -80 °C until use. After all samples were collected, target protein levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Beijing Xinbosheng Biotechnology Co., Ltd.) following the manufacturer’s instructions.

The human monocytic cell line THP-1 was purchased from Wuhan Zishan Biotechnology Co., Ltd. (Wuhan, Hubei, China). Cells were routinely maintained in complete RPMI 1640 medium (Cat. No. 11875093, Gibco, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Cat. No. 10099141C, Gibco, USA). All cultures were incubated in a humidified atmosphere with 5% CO₂ at 37 °C. THP-1 cells were passaged every 2-3 days at a split ratio of 1:3 to 1:4 when reaching 80%-90% confluence. Only cells within passage 5 to passage 20 were used for all formal experiments to guarantee stable cellular phenotypes and experimental reproducibility.

Cell line authentication was performed via short tandem repeat (STR) profiling. The STR genotype of the THP-1 cell line was verified by the supplier (Wuhan Zishan Biotechnology Co., Ltd., China) prior to delivery. The genotyping results were fully consistent with the standard THP-1 profile in the ATCC cell bank, and no cross-contamination with other human cell lines was detected. Mycoplasma contamination was routinely screened for all cell batches using a PCR-based mycoplasma detection kit, and all cells applied in this study were confirmed to be mycoplasma-free.

For macrophage differentiation, THP-1 cells in the logarithmic growth phase were counted using an automated cell counter and seeded into 6-well plates at a density of 5 × 10⁶ cells per well, with 2.5 mL of complete culture medium added to each well. To optimize the differentiation condition, cells were treated with a gradient of phorbol 12-myristate 13-acetate (PMA, MCE, Cat. No. HY-18739) concentrations, and cell adherence status was evaluated at serial time points (0, 6, 12, 18, 24, 30, 36, and 48 h). A PMA concentration of 75 ng/mL and an induction period of 24 h were determined as optimal to differentiate THP-1 monocytes into adherent macrophage-like cells.

For BCG infection optimization, diluted BCG suspension was added to differentiated macrophages at multiplicities of infection (MOIs) of 0, 2, 4, 8, 12, 16, 20, and 24. Cell morphology, viability, and differentiation status were assessed to screen the optimal infection dose. Subsequently, time-gradient experiments (0, 6, 12, 18, 24, 30, 36, and 48 h) were performed at the optimized MOI to characterize dynamic cellular responses and determine the optimal infection duration. All incubations were conducted under the standard culture conditions described above.

Small interfering RNA (siRNA) sequences targeting LBP were designed by Shanghai Gima Pharmaceutical Biotechnology Co., Ltd. A 4 OD siRNA was synthesized for use in cell transfection experiments. Under optimized conditions of concentration and incubation time, 10 μL of siRNA was mixed with 15 μL of Zeta transfection reagent in a sterile centrifuge tube, gently vortexed, and incubated at room temperature in a laminar flow hood for 20 min to allow formation of siRNA-lipid complexes. The resulting complex solution was then added dropwise to each well of a 6-well plate, with 25 μL applied per well. The plate was gently shaken to ensure uniform distribution of the transfection mixture. Cells were subsequently incubated in a standard cell culture incubator (37 °C, 5% CO₂) for 24 h, after which the culture medium was replaced. The cells were then further incubated for an additional 24 hours before proceeding to downstream assays.

Total cellular proteins were extracted using ice-cold RIPA lysis buffer supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Servicebio, China). After lysis on ice for 30 min with intermittent vortexing, the lysates were centrifuged at 12,000 ×g for 15 min at 4 °C to collect the supernatant containing total cellular proteins. Protein concentration was determined using a BCA protein assay kit (Servicebio, China).

For each sample, 30 μg of total protein was loaded and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% (w/v) skim milk powder dissolved in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature, and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: LBP, TLR2 and TLR4 (Servicebio, China, 1:1,000); TLR9, NF-κB p65, myeloid differentiation primary response 88 (MyD88), NLR family pyrin domain containing 3 (NLRP3), Caspase-1, C-C motif chemokine ligand (CCL3), and IL-1β (Affinity, China, 1:1,000); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Affinity, China, 1:5,000).

After five washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Affinity, China, 1:10,000) for 1 h at room temperature. After another five washes with TBST, an enhanced chemiluminescence (ECL) reagent was added for band visualization. Finally, ImageJ software was used to quantify the grayscale values of the target bands, and relative protein expression levels were normalized to the internal reference GAPDH for comparative analysis.

All statistical analyses were performed using SPSS 26.0 software. Quantitative data are presented as mean ± standard deviation (mean ± SD). Biological replicates refer to independent experiments performed on biologically distinct samples. All sample sizes (denoted as n) in this study represent the number of biological replicates. Technical replicates refer to repeated measurements of the same biological sample. The average value of technical replicates was calculated as the final value for each corresponding biological replicate, and technical replicates were not included in the statistical sample size.

Prior to all parametric analyses, the Shapiro-Wilk test was used to verify the normality of data distribution, and Levene’s test was used to assess the homogeneity of variances across groups. All datasets in this study conformed to the assumptions of normal distribution and equal variance; thus, parametric statistical methods were adopted. For two-group comparisons (including the proteomic screening with 3 samples per group, as well as qRT-PCR and ELISA validation with 40 clinical samples per group), differences between the STB group and healthy control group were analyzed by two-tailed unpaired Student’s t-test. For multi-group comparisons (including the time-gradient and concentration-gradient optimization of BCG infection, and the four-group cell intervention experiment), overall differences among groups were analyzed by one-way analysis of variance (one-way ANOVA), and Tukey’s post-hoc test was applied for pairwise multiple comparison correction. A P-value < 0.05 was considered statistically significant.

Using iTRAQ combined with LC-MS/MS, the plasma protein expression profiles of patients with STB and healthy controls were compared. Differentially expressed proteins (DEPs) were screened using the criteria of fold change (FC) > 1.20 or < 0.83 and P < 0.05. A total of 16 DEPs were identified between the two groups, including 11 upregulated proteins and 5 downregulated proteins in the STB group [Figure 1A and B]. The overall distribution of DEPs was visualized by volcano plot analysis, and hierarchical clustering analysis further demonstrated distinct protein expression patterns between the STB group and the healthy control group.

To comprehensively verify the differential expression of all screened proteins, all 16 DEPs were subjected to qRT-PCR validation at the mRNA level. Based on the magnitude of differential expression, statistical significance, and potential biological relevance to STB pathogenesis, 10 core candidate proteins closely associated with innate immunity, inflammatory regulation and tissue remodeling were further selected for protein-level validation by ELISA and subsequent mechanistic exploration. These core candidate proteins included LBP, S100 calcium-binding protein A8/A9 (S100A8/A9), serum amyloid A2 (SAA2), leucine-rich alpha-2-glycoprotein 2 (LRG2), proteoglycan 4 (PRG4), complement factor I (CFI), serpin family A member 1 (SERPINA1), cathepsin D (CTSD), ceruloplasmin (CP), and vinculin (VCL) [Figure 1C and D].

qRT-PCR of peripheral blood gene expression in patients with STB and healthy controls revealed that the LBP and S100A8/A9 genes were significantly overexpressed (P < 0.05). In addition, the genes SAA2, LRG2, PRG4, CFI, SERPINA1, CTSD, CP, VCL, IGLV4-60, IGHV3-64D, IGKV2D-29, IGKV6D-41, and IGHV1-45 showed either upregulation or downregulation in peripheral blood samples from STB patients; however, these changes did not reach statistical significance (P > 0.05, n = 40 subjects per group) [Table 2].

The proteins were quantified in patients with STB using commercial ELISA kits. The results demonstrated that plasma levels of LBP and S100-A8/S100-A9 in STB patients were significantly elevated compared to controls, and their protein expression showed a positive correlation with corresponding gene expression at the transcriptional level. In contrast, no significant differences in protein levels were observed for SAA2, LRG2, PRG4, CFI, SERPINA1, CTSD, CP, or VCL between STB patients and healthy controls, and the expression of these proteins did not correlate with their respective mRNA levels. These discrepancies may reflect post-transcriptional regulation, secretion dynamics, protein stability, or differences between cellular transcription and circulating protein abundance [Figure 2].

KEGG pathway enrichment analysis identified several significantly enriched signaling pathways related to the differentially expressed proteins [Table 3]. Notably, LBP was annotated to the TLR and NF-κB signaling pathways, which were identified as significantly enriched pathways among the differentially expressed proteins. These findings suggest that the identified differentially expressed proteins and their associated signaling pathways may play critical roles in the pathogenesis of STB.

To delineate the functional signaling pathways modulated by BCG stimulation and LBP knockdown in macrophages, KEGG pathway enrichment analysis was performed on differentially expressed proteins derived from each of the four THP-1 macrophage treatment groups, and the corresponding enrichment profiles are shown in Figure 3A-D. The results demonstrated that the differentially expressed proteins under different intervention conditions were predominantly associated with multiple innate immune and inflammatory-related pathways, including the Toll-like receptor signaling pathway, TNF signaling pathway, and IL-17 signaling pathway.

Notably, the Toll-like receptor signaling pathway and its downstream NF-κB signaling cascade were consistently identified as core enriched pathways, which were fully consistent with the pathway annotation of LBP, the key differential molecule screened from the plasma proteomics of STB patients. This concordance between clinical sample profiling and in vitro cellular experiments further suggests that these differential proteins and their related signaling pathways may participate in the inflammatory response and pathological progression of STB.

Under the conditions of inducing THP-1 monocytes into macrophages, concentration and time gradients were systematically evaluated. Based on prior studies, differentiation was assessed at time points of 0, 6, 12, 18, 24, 30, 36, and 48 hours. The MOI gradients tested were 0, 2, 4, 8, 12, 16, 20, and 24. The results demonstrated that the optimal incubation time for BCG infection was 24 h, with an optimal concentration of 8 MOI [Figure 4].

To further investigate the potential association between LBP and TLR-related inflammatory signaling, the expression levels of TLR2, TLR4, and TLR9 were examined in THP-1-derived macrophages under different treatment conditions. Compared with the control group, the expression of LBP was increased in BCG-stimulated macrophages. Under the same conditions, TLR4 expression was also elevated, whereas no obvious changes were observed in TLR2 or TLR9 expression.

Following siRNA-mediated knockdown of LBP, the protein expression levels of LBP and TLR4 were reduced in BCG-stimulated macrophages compared with those in the BCG group. In contrast, the expression levels of TLR2 and TLR9 showed no marked changes after LBP knockdown. These findings suggest that LBP may be associated with TLR4-related signaling changes in BCG-stimulated macrophages [Figure 5].

To further assess the association between LBP and NF-κB inflammatory signaling, the total protein expression level of NF-κB p65 was examined in THP-1-derived macrophages under different treatment conditions. Compared with the control group, BCG stimulation increased the total protein levels of LBP, TLR4, and NF-κB p65. Following siRNA-mediated knockdown of LBP, the total NF-κB p65 protein level was reduced in BCG-stimulated macrophages compared with the BCG-only group.

These findings suggest that LBP knockdown is associated with attenuated NF-κB-related signaling changes in BCG-stimulated macrophages, indicating that LBP may be involved in the regulation of inflammatory signaling under mycobacterial infection conditions [Figure 6].

To further evaluate the effects of LBP on inflammatory mediator expression, the protein levels of NLRP3, MyD88, Caspase-1, interleukin-1β (IL-1β), and CCL3 were examined in THP-1-derived macrophages under different treatment conditions. Compared with the control group, BCG stimulation increased the expression of NLRP3, Caspase-1, and IL-1β [Figure 7]. After siRNA-mediated knockdown of LBP, the expression levels of these inflammatory mediators were reduced in BCG-stimulated macrophages compared with those in the BCG group. For the signaling adaptor protein MyD88, no statistically significant difference in total protein expression was observed between the BCG-stimulated group and the blank control group. Compared with the BCG+siRNA group, the Con+siRNA group showed an upregulated baseline level of MyD88 protein.

In addition, CCL3 expression was altered under BCG stimulation and following LBP knockdown. Taken together, these findings suggest that LBP may be involved in the regulation of inflammatory mediator expression in BCG-stimulated macrophages [Figure 8].