Abstract: BACKGROUND: Studies have shown that there is a close association between spinal cord injury and ferroptosis, and that tetramethylpyrazine has the function of regulating redox reactions. 
OBJECTIVE: To investigate the regulatory effect of tetramethylpyrazine on ferroptosis in rats with spinal cord injury and its mechanism.
METHODS: Thirty-six female specific pathogen-free Sprague-Dawley rats were randomly divided into sham-operated group, model group and tetramethylpyrazine group, with 12 rats in each group. Animal models of spinal cord injury were established using the modified Allen’s method in the latter two groups. No treatment was given in the sham-operated group, while rats in the model and tetramethylpyrazine groups were given intraperitoneal injection of normal saline and tetramethylpyrazine solution, once a day, for 28 days.
RESULTS AND CONCLUSION: The Basso, Beattie & Bresnahan Locomotor Rating Scale score in the tetramethylpyrazine group was lower than that in the sham-operated group but higher than that in the model group after 14, 21, and 28 days of treatment (P < 0.05). After 28 days of treatment, hematoxylin-eosin staining showed that in the model group, the spinal cord tissue of rats showed cavity formation, necrotic tissue and inflammatory infiltration with fibrous tissue formation; in the tetramethylpyrazine group, the area of spinal cord tissue defects was smaller, and inflammatory infiltration and fibrous tissue formation were less than those in the model group. After 28 days of treatment, Prussian blue staining showed that a large amount of iron deposition was seen in the spinal cord tissue of rats in the model group, and less iron deposition was seen in the spinal cord tissue of rats in the tetramethylpyrazine group than in the model group. After 28 days of treatment, the levels of glutathione and superoxide dismutase in the rat spinal cord tissue were decreased (P < 0.05) and the level of malondialdehyde was increased in the model group compared with the sham-operated group (P < 0.05); the levels of glutathione and superoxide dismutase in the rat spinal cord tissue were increased (P < 0.05) and the level of malondialdehyde was decreased in the tetramethylpyrazine group compared with the model group (P < 0.05). After 28 days of treatment, qRT-PCR and western blot assay showed that the mRNA and protein levels of glutathione peroxidase 4, ferritin heavy chain, and ferroportin in the rat spinal cord tissue in the model group were decreased compared with those in the sham-operated group (P < 0.05), while the mRNA and protein levels of glutathione peroxidase 4, ferritin heavy chain, and ferroportin in the rat spinal cord tissue in the tetramethylpyrazine group were increased compared with those in the model group (P < 0.05). Immunofluorescence staining showed that after 28 days of treatment, the neuronal nuclei positive staining in the spinal cord of rats was the most in the sham-operated group and the least in the model group. To conclude, tetramethylpyrazine can improve motor function and play a neuroprotective role in rats with spinal cord injury by regulating ferroptosis.

Key words: tetramethylpyrazine, spinal cord injury, ferroptosis, neuroprotective effect, oxidative stress