Abstract:

Objective To investigate the effect of stromal cell-derived factor 1 （SDF-1） on ferroptosis of Schwann cells （SCs） after peripheral nerve injury （PNI）， and to elucidate its possible mechanism. Methods Animal experiment， 45 male 8-week-old SD rats were randomly divided into sham operation group， PNI group and SDF-1 treatment group （PNI+SDF-1 group）， with 15 rats in each group. The rats in sham operation group were subjected to sciatic nerve exposure without injury； the rats in PNI group and SDF-1 treatment group were subjected to sciatic nerve clamping method to establish the models. After modeling， the rats in PNI group were intramuscularly injected with 0.2 mL·d^-1 normal saline， and the rats in SDF-1 treatment group were intramuscularly injected with 4 μg·kg^-1·d^-1 SDF-1 for 5 consecutive days. On the 5th day after operation， 10 rats from each group were randomly selected and sacrificed to harvest the sciatic nerves. Western blotting method was used to detect the expression levels of long-chain acyl-CoA synthetase 4 （ACSL4）， glutathione peroxidase 4 （GPX4）， and ferroptosis suppressor protein 1 （FSP1） proteins in sciatic nerve of the rats in various groups； transmission electron microscope was used to observe the morphology of mitochondria in SCs in the sciatic nerve； Western blotting method was used to detect the expression levels of extracellular signal-regulated kinase （ERK）， phosphorylated extracellular signal-regulated kinase （p-ERK）， and nuclear factor erythroid 2-related factor 2 （Nrf2） proteins in the sciatic nerve. On the 14th day after operation， the remaining 5 rats from each group were sacrificed to harvest the sciatic nerves. HE staining was used to observe the morphology of sciatic nerve fibers； immunofluorescence staining was used to detect the expression levels of neurofilament 200 （NF200） and myelin basic protein （MBP） in sciatic nerve of the rats in various groups. Cell experiment， the SCs were divided into control group， lipopolysaccharide （LPS）- induced injury group （LPS group） and SDF-1 treatment group （LPS+SDF-1 group）. 2'，7'- Dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence probe method was used to detect the reactive oxygen species （ROS） levels in the SCs in various groups； FeRhoNox-1 fluorescence probe method was used to detect the ferrous ion （Fe²⁺）） levels in the SCs in various groups； immunofluorescence staining was used to detect the expression of ACSL4 and GPX4 proteins in the SCs in various groups； Western blotting method was used to detect the protein expression levels of ACSL4， GPX4， FSP1， ERK， p-ERK， and Nrf2 in the SCs in various groups. Rescue experiment， the SCs were divided into LPS group， LPS+SDF-1 group， LPS+SDF-1+SCH772984 group and LPS+SCH772984 group； Western blotting method was used to detect the expression levels of Nrf2， ACSL4， GPX4， and FSP1 proteins in the SCs in various groups. Results In animal experiment， on the 5th day after operation， compared with sham operation group， the expression level of ACSL4 protein in sciatic nerve of the rats in PNI group was increased （P<0.01）， while the protein expression levels of GPX4 and FSP1 proteins were decreased （P<0.05）； the mitochondria in SCs were shrunken and the cristae structure was blurred； the p-ERK/ERK ratio and the protein expression level of Nrf2 were decreased （P<0.01）. Compared with PNI group， the expression level of ACSL4 protein in sciatic nerve of the rats in PNI+SDF-1 group was decreased （P<0.05）， while the expression levels of GPX4 and FSP1 proteins were increased （P<0.05）； the mitochondria in SCs showed increased volume and cristae structure tended to be normal； the p-ERK/ERK ratio and the protein expression level of Nrf2 were increased （P<0.01）. The HE staining results showed that on the 14th day after operation， compared with sham operation group， the sciatic nerve fibers in PNI group were arranged in disorder， and the fluorescence intensities of NF200 and MBP proteins in the sciatic nerve were decreased （P<0.01）； compared with PNI group， the sciatic nerve fiber arrangement in PNI+SDF-1 group tended to be normal， and the expression levels of NF200 and MBP proteins in the sciatic nerve were increased （P<0.05）. In cell experiment， compared with control group， the levels of ROS and Fe²⁺ in the SCs in LPS group were increased （P<0.01）， the expression level of ACSL4 protein was increased （P<0.01）， the expression level of GPX4 protein was decreased （P<0.05 or P<0.01）， the expression level of FSP1 protein was decreased （P<0.01）， and the p-ERK/ERK ratio and Nrf2 protein expression level were decreased （P<0.01）. Compared with LPS group， the levels of ROS and Fe²⁺ in the SCs in LPS+SDF-1 group were decreased （P<0.05）， the expression level of ACSL4 protein was decreased （P<0.05）， the expression level of GPX4 protein was increased （P<0.05）， the expression level of FSP1 protein was increased （P<0.05）， and the p-ERK/ERK ratio and Nrf2 protein expression level were increased （P<0.01）. In rescue experiment， compared with LPS group， the expression level of Nrf2 protein in the SCs in LPS+SDF-1 group was increased （P<0.01）， the expression level of ACSL4 protein was decreased （P<0.05）， and the expression levels of GPX4 and FSP1 proteins were increased （P<0.01）； the expression level of Nrf2 protein in the SCs in LPS+SCH772984 group was decreased （P<0.05）， the expression level of ACSL4 protein was increased （P<0.05）， and the expression levels of GPX4 and FSP1 proteins were decreased （P<0.05）. Compared with LPS+SDF-1 group， the expression level of Nrf2 protein in the SCs in LPS+SDF-1+SCH772984 group was decreased （P<0.01）， the expression level of ACSL4 protein was increased（P<0.05）， and the expression levels of GPX4 and FSP1 proteins were decreased（P<0.05）. Conclusion SDF-1 can inhibit ferroptosis of the SCs after PNI and promote the repair of the injured sciatic nerve， and its mechanism is related to the activation of ERK/Nrf2 signaling pathway.

Key words: Nerve regeneration, Stromal cell-derived factor 1, Schwann cells, Ferroptosis, Extracellular signal-regulated kinase