[Screening and in vivo validation of key targets for triclosan-associated intervertebral disc degeneration]

Intervertebral disc degeneration (IVDD) is an important pathological basis of chronic low back pain and various degenerative spinal disorders, and exposure to environmental pollutants may represent a potential exogenous factor affecting intervertebral disc homeostasis. Triclosan (TCS) is a widespread environmental antimicrobial pollutant, but its molecular relationship with IVDD remains unclear. This study aims to investigate the potential molecular links between TCS and IVDD, identify candidate molecules associated with TCS exposure-related IVDD, and validate them in vivo using animal experiments.

Six IVDD transcriptomic datasets were obtained from the Gene Expression Omnibus (GEO) database. GSE23130, GSE56081, and GSE124272 constituted the training cohort, whereas GSE15227, GSE34095, and GSE70362 constituted the external validation cohort. Surrogate variable analysis (SVA) combined with the ComBat algorithm was used to correct for batch effects, and Limma was used to identify differentially expressed genes in IVDD. Potential TCS targets were predicted by integrating the ChEMBL, PharmMapper, and SwissTargetPrediction databases. Shared candidate genes between TCS and IVDD were identified and subjected to protein-protein interaction network analysis and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Ten machine-learning algorithms and their combinations were used to construct 127 predictive models. External validation together with Shapley additive explanation (SHAP) analysis was used to prioritize candidate molecules, and AutoDock Vina was used to evaluate the potential binding affinity of TCS for candidate proteins. For in vivo experiments, 8-week-old C57BL/6J mice were randomly assigned to a control group or a TCS-exposure group. Mice in the TCS group received TCS orally through the diet at 200 mg TCS/kg diet (200 ppm) for 12 consecutive weeks. Micro-computed tomography was used to measure the mean L_4/5 intervertebral disc height. Safranin O-Fast Green staining was used to evaluate nucleus pulposus cell number, intervertebral disc architecture, proteoglycan staining, and histological degeneration scores. Immunohistochemistry and Western blotting were used to assess the expression of arachidonate 5-lipoxygenase (ALOX5) and peroxisome proliferator-activated receptor delta (PPARD) in intervertebral disc tissues.

A total of 238 candidate targets of TCS were identified, and 9 shared TCS-IVDD candidate genes were obtained: HMGCR, PPARD, NR1H2, SEC14L2, FGFR1, ALOX5, LTA4H, ACSS2, and NQO2. Enrichment analyses showed that these genes were mainly involved in lipid and steroid metabolism, arachidonic acid metabolism, and apoptotic cell clearance. External validation showed that PPARD, FGFR1, and SEC14L2 were downregulated in IVDD tissues, whereas ALOX5, HMGCR, NR1H2, LTA4H, and NQO2 were upregulated (all P<0.05). SHAP analysis showed that PPARD and ALOX5 made relatively high contributions to model prediction. Molecular docking showed that the minimum binding energies of TCS with ALOX5, PPARD, FGFR1, and HMGCR were -7.8, -7.2, -7.2, and -6.1 kcal/mol, respectively. Animal experiments showed that, compared with the control group, mice in the TCS group had a significantly lower mean L_4/5 intervertebral disc height [(272.32±18.89) μm vs (206.50±13.15) μm], fewer nucleus pulposus cells (618.20±36.46 vs 377.20±23.37), and a significantly higher histological degeneration score (2.60±1.14 vs 12.20±1.48) (all P<0.001). Safranin O-Fast Green staining showed blurred boundaries between the nucleus pulposus and annulus fibrosus, disorganized tissue architecture, and reduced proteoglycan staining in the TCS group. Immunohistochemistry showed increased ALOX5-positive expression and decreased PPARD-positive expression (both P<0.001). Western blotting showed increased relative expression of ALOX5 and decreased relative expression of PPARD (both P<0.05).

Under a dietary exposure regimen of 200 mg TCS/kg diet for 12 consecutive weeks, TCS induced an IVDD phenotype in mice. Decreased PPARD and increased ALOX5 expression were consistent with TCS exposure-related pathological changes in the intervertebral disc, suggesting that dysregulation of lipid metabolism and inflammatory lipid mediators may be involved in this process. PPARD and ALOX5 may represent key candidate molecules linking TCS exposure, lipid metabolic dysregulation, and IVDD.