SARS-CoV-2 Spike Peptides Trigger Nociceptive Responses Through Spinal TLR4 Pathways

COVID-19 has affected over 700 million people worldwide, with a significant portion of the population experiencing severe pulmonary and circulatory complications, often accompanied by symptoms such as prostration and pain. Moreover, the manifestation of these symptoms and other COVID-19-related complications varies depending on viral mutations, particularly those occurring in the spike (S) protein of SARS-CoV-2. Therefore, the present study aimed to investigate the effects of three different S proteins on nociceptive threshold, as well as the spinal involvement of TLR4 and microglia in this process. Male C57BL/6 mice received intrathecal administration of three synthetic peptides (PSPD2001, PSPD2002 and PSPD2003) derived from the SARS-CoV-2 S protein or saline. Nociceptive threshold was assessed using the von Frey filament test before and after peptide administration. The spinal involvement of Toll-like receptor 4 (TLR4), microglia, p38 MAPK, and NF-κB was evaluated using specific antagonists and inhibitors. mRNA expression of TLR4 was assessed by RT-PCR, pro-inflammatory cytokine levels by ELISA, and microglial activation in the dorsal horn of the spinal cord was analyzed by immunofluorescence in wild-type, CX3CR1 ^GFP□/□ , and TLR4 ^□/□ mice. In addition, molecular dynamics analysis was performed to assess the temporal stability of the PSPD2003–TLR4 complex. Pharmacological data demonstrated that the peptides induced nociception involving TLR4, microglia, p38 MAPK, and NF-κB. Notably, PSPD2003 increased TLR4 mRNA expression and elevated TNF-α and IL-6 levels in the spinal cord. PSPD2003 also enhanced microglial activation in the spinal cord, which was abolished in TLR4□/□ mice. Molecular dynamics analysis results robustly demonstrate that PSPD2003 forms a stable and functionally relevant complex with TLR4. These findings suggest that SARS-CoV-2 S protein-derived peptides contribute to pain during COVID-19 infection, with spinal TLR4 and microglia playing key roles in this process.