Abstract: BACKGROUND: The clinical management of spinal cord injury remains a global medical challenge, with no currently available ideal treatment. Traditional Chinese medicine has therapeutic advantages for spinal cord injury. Notably, tetramethylpyrazine, an active component of Chuanxiong rhizome, has been shown to significantly suppress pathological responses including neuroinflammation and apoptosis after spinal cord injury, exhibiting promising therapeutic potential. However, its mechanisms require further elucidation.
OBJECTIVE: To investigate the regulatory effects of tetramethylpyrazine on the Kelch-like ECH-associated protein 1/nuclear factor-erythroid 2-related factor 2 (Keap-1/Nrf2) signaling pathway and iron metabolism following spinal cord injury, and to elucidate its neuroprotective mechanisms.
METHODS: Thirty-six Sprague-Dawley rats were randomly allocated into: sham group (laminectomy+saline, n=12), model group (spinal cord injury+saline, n=12), and tetramethylpyrazine group (spinal cord injury+tetramethylpyrazine, n=12). At 4 weeks post-injury, tissues were collected for analysis. Neuronal morphology was assessed via Nissl staining; iron deposition detected by Prussian blue staining; iron content in the spinal cord measured using an iron assay kit; protein expression of Keap-1, ferritin heavy polypeptide 1, and ferritin light chain detected by immunohistochemistry; Nrf2 expression detected via immunofluorescence; and protein levels of Nrf2, ferritin heavy polypeptide 1, and ferritin light chain measured using western blot assay. RT-PCR was used to quantify the mRNA expression of Nrf2, Keap-1, ferritin light chain, and ferritin heavy polypeptide 1.
RESULTS AND CONCLUSION: Nissl staining demonstrated preserved neuronal morphology in the sham group, while the model group exhibited disorder of neuronal structure and tetramethylpyrazine administration substantially improved these pathological morphological changes. Quantitative assessment of iron metabolism showed that both the model and tetramethylpyrazine groups displayed significantly elevated average absorbance values for Prussian blue staining (P < 0.01) and iron content in the spinal cord tissue (P < 0.01) compared with the sham group, while the iron content in the tetramethylpyrazine group was significantly lower than that in the model group (P < 0.01). Immunohistochemical analysis revealed that the model and tetramethylpyrazine groups showing markedly increased average absorbance values for Keap-1 expression but decreased average absorbance values for ferritin light chain and ferritin heavy chain 1 levels compared with the sham group (all P < 0.01). Importantly, tetramethylpyrazine treatment reversed these patterns, demonstrating significantly lower average absorbance values for Keap-1 and higher average absorbance values for ferritin light chain/ferritin heavy chain 1 expression compared with the model group (P < 0.01). Immunofluorescence results showed that the average absorbance values for Nrf2 expression in the model and tetramethylpyrazine groups were significantly lower than those in the sham group (P < 0.01), while the average absorbance value for Nrf2 expression in the tetramethylpyrazine group was significantly higher than that in the model group (P < 0.01). Western blot analysis confirmed these trends at the protein level, showing decreased relative protein expression of Nrf2, ferritin heavy chain 1 and ferritin light chain in the model and tetramethylpyrazine groups compared with the sham group (P < 0.01) but increased relative protein expression of Nrf2, ferritin heavy chain 1 and ferritin light chain in the tetramethylpyrazine group compared with the model group (P < 0.01). Molecular analysis via RT-PCR showed that the relative mRNA expression of Keap-1 in the model and tetramethylpyrazine groups was significantly higher than that in the sham group (P < 0.01), while the relative mRNA expression of Nrf2, ferritin heavy chain 1, and ferritin light chain mRNA was significantly lower than that in the sham group (P < 0.01); the relative mRNA expression of Keap-1 in the tetramethylpyrazine group was significantly lower than that in the model group (P < 0.01). To conclude, tetramethylpyrazine modulates the Keap-1/Nrf2 pathway and ferritin light chain/ferritin heavy chain 1 expression after spinal cord injury, ameliorating iron metabolism dysregulation and exerting neuroprotective effects.

Key words: spinal cord injury, tetramethylpyrazine, Keap-1/Nrf2 signal pathway, iron metabolism, ferroptosis, tissue-engineered construction