Whether ferroptosis specific inhibitor could promotes spinal

Whether ferroptosis specific inhibitor could promotes spinal cord repair is a question worth to explore. A potent and stable ferroptosis specific inhibitor is essential for in vivo study. Ferrostatin-1 (Fer-1), the first generation of ferroptosis inhibitor, was shown actively suppressing ferroptosis in vitro (Zilka et al., 2017). However, its in vivo functionality is weak due to plasma and metabolic instability. SRS 16-86 is the third generation small molecule ferroptosis specific inhibitor that inhibits lipid ROS. It is more stable and potent (Linkermann et al., 2014), and was reported to strongly suppress ferroptosis in ischemia-reperfusion injury in acute renal failure.
Results
Discussion Ferroptosis is a newly discovered cell death mechanism that has been demonstrated in the Parkinson’s disease and stroke (Do Van et al., 2016, Guiney et al., 2017, Zille et al., 2017). However, its role in SCI has not been reported. In this report we found that upon SCI, the key regulators of ferroptosis such as GPX4, GSH and xCT were downregulated. The 4HNE representing the lipid peroxidation level is upregulated. By analyzing the levels of neuronal death and functional outcomes after inhibition of ferroptosis with SRS 16-86, we found that increased neuronal survival and improved locomotor recovery by ferroptosis inhibition. The gliosis and dextromethorphan hydrobromide synthesis level of inflammatory cytokines were also decreased upon treatment of SRS 16-86. The beneficial effect of ferroptosis interference opens a new pathway to reduce cell death and promote SCI repair. SRS 16-86 is a newly generated ferroptosis inhibitor which is more stable and potent than the first-in-class compound, ferrostain-1 (Fer-1). It exerted strong protection in ischemia–reperfusion injury of kidney (Linkermann et al., 2014). Here in SCI model, SRS 16-86 elevated GSH concentrations and decreased the lipid ROS marker 4HNE in spinal cord tissue. The GPX4 and xCT are ferroptosis markers, and their expression were down regulated upon injury and increased with ferroptosis inhibitor treatment. These results suggest that SRS 16-86 interfere with the ferroptosis process. The beneficial effects of ferroptosis inhibitor also include decreased expression of GFAP positive cells, indicating decreased gliosis. HE staining showed more preserved tissue upon ferroptosis inhibitor treatment. In the acute phase of SCI, blood–brain barrier (BBB) is disrupted so that drugs could get into the spinal cord tissue (Lee et al., 2012). However, our preliminary analysis did not indicate that SRS 16-86 could penetrate the intact blood–brain barrier (BBB) in rats. We established the measurement method using HPLC (Fig. S3). In order to test the BBB penetration ability of SRS 16-86, 15 mg/kg SRS 16-86 was given intravenously and after 30 min and 1 h, the cerebral spinal fluid, brain tissue homogenate and serum sample was collected. The samples were collected in triplicate, n = 3 animals. Although the serum concentration of SRS 16-86 could be detected (684.6667 ± 107.6 ppb, 1 h post injection, 1 ppb = 1 μg/L), the CSF and brain tissue homogenate concentrations were below the detection level (detection level = 16.57 ppb) (unpublished results). This is our preliminary examination of the BBB penetration ability of SRS 16-86. Modification of this compound chemical structure or drug delivery method to improve the BBB penetration ability in the future development in CNS phamacology. SRS 16-86 treatment lowered the expression of pro-inflammatory cytokines IL1β, TNF-α and ICAM-1. This indicated that ferroptosis inhibition may also results in prohibition of inflammatory cascade in the SCI secondary phase. Lipid peroxidation in ferroptosis may generate signaling molecules of inflammation. This result is consistent with the effect of Fer-1 which reduced pro-inflammatory cytokines in acute kidney injury model (Linkermann et al., 2014). Whether ferroptosis accounts for the inflammatory microenvironment of SCI is a question needed further study.