[Lobetyolin inhibits T-BHP-induced ferroptosis in human nucleus pulposus cells by regulating PI3K/AKT pathway]

This study aimed to investigate the effect of lobetyolin(LOB) on tert-butyl hydroperoxide(T-BHP)-induced ferroptosis in human nucleus pulposus cells(NPCs) based on the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT) pathway, thereby providing a theoretical basis for the treatment of intervertebral disc degeneration(IDD). Human NPCs were cultured in vitro. The cell counting kit-8(CCK-8) assay was used to screen the optimal concentration for T-BHP induction and the optimal intervention concentration of LOB. Cells in the logarithmic growth phase were randomly divided into a blank control group, a model group, a Ferrostatin-1(Fer-1) control group, a model + Fer-1 group, a LOB group, and a PI3K/AKT agonist 740Y-P group. Cell viability was assessed using the CCK-8 assay. Intracellular ferrous ion(Fe~(2+)), malondialdehyde(MDA), and glutathione(GSH) levels were measured using colorimetric assays. Mitochondrial ultrastructure was observed using transmission electron microscopy(TEM). The protein expression levels of solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4), collagen type Ⅱ(collagen Ⅱ), matrix metallopeptidase-13(MMP-13), phosphorylated phosphatidylinositol 3-kinase(p-PI3K), and phosphorylated protein kinase B(p-AKT) were determined by Western blot. Additionally, the expression levels of p-PI3K and p-AKT were detected by immunofluorescence. Compared with that in the blank control group, the viability of human NPCs in the model group decreased significantly. Intracellular Fe~(2+) and lipid peroxidation product MDA levels increased significantly, while the antioxidant indicator GSH level decreased significantly in the model group. Protein analysis showed that the expression of SLC7A11, GPX4, and collagen Ⅱ was significantly downregulated, while MMP-13 expression was upregulated. The expression levels of p-PI3K and p-AKT were inhibited. Typical morphological changes of ferroptosis, such as mitochondrial shrinkage and reduction of cristae, were observed under a transmission electron microscope. Compared with the model group, the LOB group, model + Fer-1 group, and 740Y-P group showed significantly reduced levels of Fe~(2+) and MDA, increased GSH levels, upregulated protein expression of SLC7A11, GPX4, and collagen Ⅱ, and downregulated MMP-13 expression. Furthermore, LOB intervention significantly upregulated the expression levels of p-PI3K and p-AKT in human NPCs, and the improvement trend of related indicators was consistent with that of the 740Y-P group. LOB effectively alleviates T-BHP-induced ferroptosis in human NPCs and reduces extracellular matrix degradation. Its protective mechanism may be achieved by regulating the PI3K/AKT signaling pathway.