Tuina Analgesia Is Associated With the Modulation of the NCOA4-Mediated Ferroautophagy-Ferroptosis Pathway in SNL-Induced Neuropathic Pain Rats.

Abstract

BACKGROUND: Neuropathic pain (NP) has become a major global public health issue. Tuina, a traditional therapy, has been shown to alleviate pain effectively by promoting nerve repair and reducing neuroinflammation. This study aimed to investigate whether Tuina can suppress the ferroptosis-autophagy pathway to improve NP induced by spinal nerve ligation (SNL) in rats. METHODS: A total of 42 Sprague-Dawley rats were randomly allocated into seven experimental groups, with six rats in each group: Sham, SNL model, SNL&#x2009;+&#x2009;Sham Tuina, SNL&#x2009;+&#x2009;Tuina (acupoints GB30/GB34/GB39, 5&#x2009;N pressure, 120&#x2009;times/min, 18&#x2009;min/day for 14&#x2009;days), SNL&#x2009;+&#x2009;si-NC (empty vector), SNL&#x2009;+&#x2009;si-NCOA4 (NCOA4 silencing), and SNL&#x2009;+&#x2009;ferrostatin-1 (10&#x2009;mg/kg for 7&#x2009;days). Pain behaviors were assessed using paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). Protein expressions of NCOA4, ferritin heavy chain 1 (FTH1), microtubule-associated protein 1 light chain 3 (LC3), glutathione peroxidase 4 (GPX4), and acyl-CoA synthetase long-chain family member 4 (ACSL4) were detected by the western blot. Immunofluorescence visualized NCOA4-FTH1 colocalization. The mitochondrial ultrastructure was analyzed by TEM. Feand MDA levels were measured colorimetrically. RESULTS: Compared with the SNL&#x2009;+&#x2009;Sham group, the Tuina group exhibited a significantly increased PWT (p < 0.001) and PWL (p < 0.001). IHC analysis revealed that the Tuina intervention reduced the proportion of NCOA4-positive cells (p < 0.001), increased GPX4 expression (p < 0.001), and decreased ACSL4 expression (p < 0.001) in the spinal dorsal horn. Western blot further confirmed that Tuina downregulated NCOA4, LC3, and ACSL4 protein levels (all p < 0.001), while upregulating FTH1 and GPX4 (both p < 0.001). IF demonstrated enhanced colocalization of NCOA4 and FTH1 in the Tuina group (p < 0.001). TEM observations indicated that Tuina ameliorated ferroptosis-associated mitochondrial damage, including cristae disruption and membrane rupture. Biochemical assays showed that Tuina significantly reduced the spinal cord Fecontent (p < 0.001) and MDA levels (p < 0.001). CONCLUSION: Tuina alleviates NP in SNL rats, an effect correlated with the inhibition of NCOA4-mediated ferritinophagy, decreased iron overload and lipid peroxidation, and attenuated ferroptosis in spinal dorsal horn neurons.