Abstract
Background
Immune cell infiltration has been found to be strongly associated with the occurrence and progression of intervertebral disc degeneration (IDD), and intervertebral discs (IVDs) are immune-privileged organs. This research aimed to identify novel targets for therapeutic intervention and investigate significant physiological and cellular mechanisms involved in the immune infiltration processes of IDD.
Results
CIBERSORT deconvolution analysis revealed that the levels of follicular helper T cells and M2 macrophages were significantly increased during IDD, while the levels of resting mast cells were significantly decreased. WGCNA revealed that the blue module was associated with macrophage infiltration (positive correlation 0.87, P < 0.001), of which genes, including VAMP8, TLR4, JUN, PLA2G4A, NMI, RORA, IFNGR1, IFNGR2, NINJ1, TNIP1, and CD81, were mainly enriched in macrophage activation, myeloid leukocyte activation and the Toll-like receptor 4 signaling pathway. The proportion of leukocytes in the IVD was greatest on the 14th day after puncture. Single-cell sequencing revealed the division of degenerative IVD cells into seven major cell types, annulus fibrosus cells, smooth muscle cells, fibroblasts, macrophages, monocytes, vascular endothelial cells, and NP cells, in which Jun, Cd81, Rora, Vamp8, Ninj1, and Tnip1 were strongly expressed, western blotting results in normal and degenerated nucleus pulposus cells (NNP and DNP) showed that the expression of VAMP8 and c-Jun increased after 24 hours of stimulation with 50 ng/ml TNF-α. The immunohistochemical results showed an increase in VAMP8 and c-Jun levels in the IVD of rats on the 14th day after acupuncture.
Conclusion
The 14th day after acupuncture in the IVD of rats is a crucial time point for the infiltration of immune cells. This work is the first to reveal that VAMP8 and c-Jun are linked to the infiltration of macrophages in IDD. Elevated expression of VAMP8 and c-Jun was confirmed in both DNP and punctured rat IVDs.