Quercetin targets the TNF-a/TNF pathway to activate mitochondrial autophagy to improve nucleus pulposus cell function...

The primary pathogenic mechanism of lower back pain is intervertebral disc degeneration (IVDD), and the phenotypic change of nucleus pulposus cells and matrix degradation are caused by an imbalance in the "inflammation - autophagy - fibrosis" axis. Despite Tong'an decoction's obvious therapeutic benefits, it is unclear if its principal ingredient, quercetin, controls mitochondrial autophagy and postpones IVDD by interfering with TNF signaling.

Tong'an decoction's various components were screened using the traditional Chinese medicine database, and quercetin's main targets were found. By combining transcriptome differential analysis and module analysis, IVDD core genes may be identified. These genes can then be intersected with the autophagy genes to identify autophagy-associated IVDD genes. Joint validation of core genes, additional single-cell sequencing study of cell subpopulation dynamics, and external data validation of core genes expression. The in vivo effectiveness of quercetin was confirmed by imaging and pathological tissue staining, acupuncture rat models, the identification of inflammatory markers, and the RNA detection of important genes. The mechanism of action of quercetin was anticipated using a quercetin target interaction network. Finally, the lipopolysaccharide induced nucleus pulposus cells model was used for molecular mechanism and functional validation.

677 IVDD related autophagy genes (such as HIF1A, TNF, BCL2, and LC3) were screened. Functional enrichment shows that these genes are significantly involved in mitochondrial autophagy, apoptosis, ferroptosis, and inflammatory signaling pathways such as TNF, MAPK, and NF-KB. External verification found that the levels of inflammatory factors IL-1 β and TNF - α were elevated in IVDD tissues, and key genes for autophagy and apoptosis were expressed. Single cell sequencing detected different states of nucleus pulposus cells, among which fibrous nucleus pulposus cells are an important pathological type in IVDD. Trajectory analysis reveals the transition of nucleus pulposus cells from steady state to fibrotic phenotype, accompanied by the secretion of inflammatory factors by macrophages. The animal model showed that the collagen arrangement in the nucleus pulposus tissue of the model group was disordered, fibrosis occurred, and the expression of inflammation, autophagy, apoptosis hub genes HIF1A, MAPK1, NFKB, CASP3, etc. was upregulated, while BCL2 was downregulated. Cell experiments have confirmed that inflammatory stimulation leads to depolarization of mitochondrial membrane potential, elevation of autophagy markers, and swelling of mitochondrial structure, which were alleviated by quercetin intervention.

The study found that TNF/IL-1β driven macrophage infiltration stimulates the NF-κB/MAPK pathway, increasing inflammation-induced mitochondrial autophagy dysregulation. Additionally, HIF-1α hypoxic stress accelerates the transition of nucleus pulposus cells into fibroNPCs. Quercetin treatment can drastically reduce TNF signaling, restore mitochondrial autophagy equilibrium, and reverse fibrosis transformation. This study provides a complete proof chain of "components targets phenotype" for treating IVDD with Tong'an Tang, establishing the groundwork for clinical translation.