[Mechanism of Duhuo Jisheng Decoction in regulating intervertebral disc degeneration via Wnt3a/YAP/ACSL4 pathway]

This study investigated the protective effects and underlying mechanisms of Duhuo Jisheng Decoction(DHJSD) against nucleus pulposus cells(NPCs) ferroptosis and intervertebral disc degeneration(IDD) in a rat model based on the Wnt family member 3a(Wnt3a)/Yes-associated protein(YAP)/acyl-CoA synthetase long-chain family member 4(ACSL4) signaling pathway. In vitro experiments: twenty-five Sprague-Dawley(SD) rats were randomly divided into five groups and administered distilled water, low, medium, or high doses of DHJSD(4.9, 9.8, and 19.6 g·kg~(-1), respectively), or celecoxib(0.02 g·kg~(-1)) via gavage for seven consecutive days. Serum was collected following administration. NPCs degeneration was induced using interleukin-1β(IL-1β), and the effects of DHJSD-containing serum on NPCs proliferation and apoptosis were investigated. Cell viability and proliferation were measured using the CCK-8 and EdU assays, while apoptosis was assessed via Annexin V-FITC/PI dual staining. To further explore the role of the Wnt3a/YAP/ACSL4 pathway, the Wnt3a/β-catenin pathway agonist SKL2001 was applied. Western blot was performed to detect the protein expression of Wnt3a, phosphorylated glycogen synthase kinase 3β(p-GSK3β), phosphorylated β-catenin(p-β-catenin), β-catenin, YAP, ACSL4, and glutathione peroxidase 4(GPX4). Immunofluorescence staining was used to evaluate the expression of β-catenin, YAP, type Ⅱ collagen(collagen Ⅱ), and matrix metalloproteinase-13(MMP-13). Malondialdehyde(MDA) content was measured using a colorimetric assay. Mitochondrial membrane potential was assessed via the JC-1 assay, and mitochondrial ultrastructure was observed via transmission electron microscopy(TEM). In the in vivo experiments, SD rats were randomly assigned to four groups: sham operation, model, DHJSD(19.6 g·kg~(-1)), and celecoxib(0.02 g·kg~(-1)). An IDD model was established via needle puncture, followed by a 21-day intervention. Magnetic resonance imaging(MRI) of the caudal vertebrae was conducted, and the degree of disc degeneration was evaluated using Pfirrmann grading. Hematoxylin-eosin(HE) and safranin O staining were used to observe histopathological changes in intervertebral disc tissue. Western blot was employed to detect the expression of Wnt3a, p-GSK3β, β-catenin, YAP, and ACSL4 in nucleus pulposus tissue. RESULTS:: showed that DHJSD-containing serum promoted NPCs proliferation, enhanced cell viability in a dose-dependent manner, and inhibited apoptosis. Compared with the IL-1β group, DHJSD-containing serum significantly downregulated the expression of Wnt3a, p-GSK3β, β-catenin, YAP, and ACSL4, while promoting GPX4 expression. It also reduced nuclear translocation of β-catenin and YAP, increased collagen Ⅱ expression, decreased MMP-13 expression, lowered MDA content, elevated the JC-1 aggregate-to-monomer fluorescence ratio, and improved mitochondrial morphology. However, these protective effects were attenuated upon SKL2001 intervention. In the IDD rat model, both DHJSD and celecoxib significantly reduced Pfirrmann grades and histological scores, and downregulated the protein expression of Wnt3a, p-GSK3β, β-catenin, YAP, and ACSL4. In conclusion, DHJSD may ameliorate IDD by regulating the Wnt3a/YAP/ACSL4 signaling pathway, thereby inhibiting ferroptosis and extracellular matrix degradation in NPCs.