党参炔苷调控PI3K/AKT通路抑制T-BHP诱导的人髓核细胞铁死亡的机制研究OA

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(Fe2+),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 Fe2+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 Fe2+and MDA,increased GSH levels,upregulated protein expression of SLC7A11,GPX4,and collagenⅡ,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.