Methyl Ferulic Acid Alleviates Neuropathic Pain by Inhibiting Nox4-induced Ferroptosis in Dorsal Root Ganglia Neurons in Rats

Neuropathic pain is a disease that has become one of the major public health problems and a global burden. Nox4-induced oxidative stress can lead to ferroptosis and neuropathic pain. Methyl ferulic acid (MFA) can inhibit the Nox4-induced oxidative stress. This study aimed to estimate whether methyl ferulic acid alleviates neuropathic pain by inhibiting the expression of Nox4 and its induction of ferroptosis. Adult male Sprague-Dawley rats were subjected to spared nerve injury (SNI) model to induce neuropathic pain. After the establishment of the model, methyl ferulic acid was given 14 days by gavage. Nox4 overexpression was induced by microinjection of the AAV-Nox4 vector. All groups measured paw mechanical withdrawal threshold (PMWT), paw thermal withdrawal latency (PTWL), and paw withdrawal cold duration (PWCD). The expression of Nox4, ACSL4, GPX4, and ROS was investigated by Western blot and immunofluorescence staining. The changes in iron content were detected by a tissue iron kit. The morphological changes in mitochondria were observed by transmission electron microscopy. In the SNI group, the paw mechanical withdrawal threshold, the paw withdrawal cold duration decreased, the paw thermal withdrawal latency did not change, the Nox4, ACSL4, ROS, and iron content increased, the GPX4 decreased, and the number of abnormal mitochondria increased. Methyl ferulic acid can increase PMWT and PWCD but does not affect PTWL. Methyl ferulic acid can inhibit Nox4 protein expression. Meanwhile, ferroptosis-related protein ACSL4 expression was decreased, GPX4 expression was increased, ROS, iron content and abnormal mitochondrial number were decreased. By overexpressing Nox4, the PMWT, PWCD, and ferroptosis of rats were more severe than those of the SNI group, but they could be reversed after treatment with methyl ferulic acid. In conclusion, methyl ferulic acid can alleviate neuropathic pain, which is related to Nox4-induced ferroptosis.