Tuina therapy alleviates neuropathic pain by modulating the CX3CL1/CX3CR1 axis to inhibit degradation of the perineuronal nets

Introduction Tuina, a traditional Chinese non-pharmacological therapy, is effective in treating neuropathic pain (NP). However, its underlying molecular mechanisms remain elusive. The degradation of perineuronal nets (PNNs) via the CX3CL1/CX3CR1 axis is a key driver of central sensitization. This study investigates whether Tuina exerts analgesia by modulating this specific neuro-immune axis to preserve PNNs integrity in a rat model of chronic compression of the dorsal root ganglion (CCD). Methods Sprague–Dawley rats were subjected to CCD surgery to establish the NP model. To validate the role of PNNs, a subset of rats received intrathecal chondroitinase ABC (chABC). To explore Tuina’s mechanism, rats were divided into Sham, Model, Tuina, CX3CR1 inhibitor (AZD8797), and Tuina + AZD8797 groups. Standardized Tuina intervention or inhibitor administration was performed from days 4 to 14 post-surgery. Pain behaviors (PWT and PWL) were evaluated. Spinal dorsal horn tissues were analyzed using transmission electron microscopy, Western blotting, immunofluorescence, and flow cytometry. Results CCD induced severe mechanical allodynia and thermal hyperalgesia, accompanied by the structural disruption of spinal PNNs—indicated by a significant loss of WFA-binding glycosaminoglycans despite unchanged total aggrecan core protein levels—and ultrastructural neuronal damage. Mechanistically, CCD upregulated the CX3CL1/CX3CR1/PI3K/AKT cascade, which was associated with increased markers of microglial phagocytosis (upregulated TREM2 and LAMP1), as well as Ca 2+ influx and c-FOS accumulation. Tuina intervention attenuated these pathological alterations, significantly restoring WFA expression and suggesting a preservation of PNNs structural integrity. Furthermore, the combined treatment (Tuina + AZD8797) yielded an enhanced analgesic and biochemical recovery compared to either monotherapy. Conclusion Tuina produces robust central analgesia by suppressing the CX3CL1/CX3CR1-PI3K/AKT signaling pathway. This suppression restores microglial phagocytic homeostasis and crucially protects the glycosaminoglycan lattice of PNNs against excessive degradation, thereby alleviating neuropathic central sensitization.