Pharmacological network analysis of the functions and mechanism of kaempferol from Du Zhong in intervertebral disc degeneration (IDD)

Senescence and apoptosis of the nucleus pulposus cells (NPCs) are essential components of the intervertebral disc degeneration (IDD) process. Senescence and anti-apoptosis treatments could be effective ways to delay or even stop disc degeneration. IDD has been treated with Eucommia ulmoides Oliver (Du Zhong, DZ) and its active ingredients. However, the roles and mechanisms of DZ in NPC apoptosis and senescence remain unclear.Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to select the main active ingredients of DZ with the threshold of oral bioavailability (OB) ​≥ ​30% and drug-likeness (DL) ​≥ ​0.2. GSE34095 contained expression profile of degenerative intervertebral disc tissues and non-degenerative intervertebral disc tissues were downloaded for different expression genes analysis. The disease targets genes of IDD were retrieved from GeneCards. The online tool Metascape was used for functional enrichment annotation analysis. The specific effects of the ingredient on IL-1β treated NPC cell proliferation, cell senescence, reactive oxygen species (ROS) accumulation and cell apoptosis were determined by CCK-8, SA-β-gal staining, flowcytometry and western blot assays.A total of 8 active compounds of DZ were found to meet the threshold of OB ​≥ ​30% and DL ​≥ ​0.2 with 4151 drug targets. After the intersection of 879 IDD disease targets obtained from GeneCards and 230 DEGs obtained from the IDD-related GSE dataset, a total of 13 hub genes overlapped. According to functional enrichment annotation analysis by Metascape, these genes showed to be dramatically enriched in AGE-RAGE signaling, proteoglycans in cancer, wound healing, transmembrane receptor protein tyrosine kinase signaling, MAPK cascades, ERK1/2 cascades, PI3K/Akt signaling pathway, skeletal system, etc. Disease association analysis by DisGeNET indicated that these genes were significantly associated with IDD, intervertebral disc disease, skeletal dysplasia, and other diseases. Active ingredients-targets-signaling pathway networks were constructed by Cytoscape, and kaempferol was identified as the hub active compound of DZ. In the IL-1β-induced IDD in vitro model, kaempferol treatment significantly improved IL-1β-induced NPC cell viability suppression and senescence. In addition, kaempferol treatment significantly attenuated IL-1β-induced ROS accumulation and apoptosis. Furthermore, kaempferol treatment partially eliminated IL-1β-induced decreases in aggrecan, collagen II, SOX9, and FN1 levels and increases in MMP3, MMP13, ADAMTS-4, and ADAMTS-5. Moreover, kaempferol treatment significantly relieved the promotive effects of IL-1β stimulation upon p38, JNK, and ERK1/2 phosphorylation. ERK1/2 inhibitor PD0325901 further enhanced the effect of kaempferol on the inhibition of ERK1/2 phosphorylation, downregulation of MMP3 and ADAMTS-4 expression, and upregulation of aggrecan and collagen II expressions.Kaempferol has been regarded as the major active compound of DZ, protecting NPCs from IL-1β-induced damages through promoting cell viability, inhibiting cell senescence and apoptosis, increasing ECM production, and decreasing ECM degradation. MAPK signaling pathway may be involved.This study provides in vitro experimental data support for the pharmacological effects of kaempferol in treating IDD, and lays a solid experimental foundation for its future clinical application in IDD treatment.