Background: Spinal cord injury (SCI) is associated with significant paralysis and high fatality. Recent research has revealed that ferroptosis participates in the pathogenesis of SCI. Astragaloside IV (AS-IV), the main active ingredient of the plant Astragalus membranaceus, has been reported to promote motor function recovery in rats with SCI. This study explored the effects of AS-IV in H 2 O 2 -treated PC12 pheochromocytoma cells.
Methods:The optimal concentration and duration of AS-IV treatment in PC12 cells was assessed using the cell counting kit 8 (CCK-8) assay. Subsequently, the SCI cell model was established in PC12 cells using H 2 O 2 . The effects of AS-IV, FIN56, and transcription factor EB (TFEB) small interfering (si)RNA on cell viability and apoptosis in the SCI model were determined using the CCK-8 assay and flow cytometry, respectively. Caspase-3 and lactate dehydrogenase (LDH) levels were measured by colorimetric assay and enzyme-linked immunosorbent assay (ELISA), respectively. Cellular reactive oxygen species (ROS) were detected by flow cytometry combined with dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. The cellular ultrastructure was analyzed by transmission electron microscopy (TEM). The ferroptosis pathwayrelated proteins were confirmed using Western blot analysis. TFEB expression was confirmed by Western blot and immunofluorescence.
Results:The optimal concentration and duration of AS-IV treatment in PC12 cells was determined to be 1.0 μM and 48 h, respectively. AS-IV markedly accelerated proliferation, suppressed apoptosis, and reduced ROS and LDH accumulation. Furthermore, AS-IV enhanced TFEB expression in H 2 O 2 -damaged PC12 cells. The effects of AS-IV on SCI were inhibited by si-TFEB, and this inhibition was further reinforced by the addition of FIN56.
Conclusions:The results of this investigation using the SCI cell model suggested that AS-IV alleviated SCI by promoting TFEB expression and subsequently mediating ferroptosis. This may represent a potential clinical treatment for SCI.