Integrated machine learning and multi-omics analysis identifies CYP1B1 as a candidate target of orientin in intervertebral disc degeneration-associated low back pain.

Objectives: Orientin is a bioactive agent isolated from natural resources successfully applied for pain control. This study was to explore candidate hub gene of orientin to intervertebral disc degeneration (IDD) -associated low back pain. Methods: This study integrated the network pharmacology analysis using orientin and IDD-associated pain, machine learning and immune infiltration analysis using GSE70362 as prediction analysis. The validation was performed via molecular docking, molecular dynamics simulation, single-cell RNA sequencing (scRNA-seq) analysis and cell experiment using human annulus fibrosus cells under IL-1β stimulation. Results: Network pharmacology analysis showed that there were 368 overlapped genes between orientin and IDD-associated low back pain. Machine learning demonstrated that Cytochrome P450 1B1 (CYP1B1) was candidate hub genes with excellent diagnostic performance (AUC = 0.95). Immune infiltration analysis showed that CYP1B1 had no correlation with infiltration levels of any immune cell subset cells after Benjamini-Hochberg false discovery rate correction. Molecular Docking showed docking energies of orientin to CYP1B1 were all smaller than -9.5 kcal/mol. The molecular dynamics simulation showed a system of orientin-CYP1B1 showed a relatively stable conformational ensemble. scRNA-seq analysis revealed tissue- and cell-type specific expression of CYP1B1, with significant upregulation in annulus fibrosus fibroblasts from diseased samples. In cell experiments, orientin alleviated the decrease in cell viability induced by IL-1β stimulation, and down-regulated CYP1B1 gene expression and CYP1B1 protein levels. Conclusion: This study showed that CYP1B1 is a potential candidate hub gene for orientin to IDD-associated low back pain.