Ligustrazine alleviates spinal cord injury-induced neuropathic pain by inhibiting the TLR4/NF-κB signaling pathway.

Abstract

Objective: To investigate the effects of ligustrazine on neuropathic pain (NPP) in rats with sciatic nerve injury and to provide new scientific insight for broadening the clinical application of ligustrazine.

Methods: Human spinal cord cell line STR cells were transfected with TLR4-mimic or mimic negative control (mimic-NC). After transfection, the STR cells were treated with different concentrations of ligustrazine (0, 0.25, 0.5, 1, 2 μm) for 24 h or 48 h. Cell proliferation was detected by MTT assay and colony formation assay. A rat model was further constructed to evaluate mechanical and cold pain sensitivity behaviors by fiber mechanical stimulation and freezing spray. The extracellular fluids of medial prefrontal cortex (mPFC) and central amygdala (CeA) were collected by intracranial dual-site simultaneous microdialysis. The contents of glutamic acid (Glu), aspartate (Asp), glycine (Gly), and γ-aminobutyric acid (GABA) in extracellular fluids were detected by HPLC.

Results: Compared to the 0 μm group, ligustrazine concentration at 0.5 μm significantly decreased the relative cell viability of STR cells and promoted the cell apoptosis rate. Ligustrazine at 0.25 μm significantly reduced the colony number of STR cells (all P<0.05). Compared to the control group, 1 μM ligustrazine significantly increased the protein expression of Bax and cleaved caspase 3 in STR cells but decreased the protein expression of Bcl-2 (all P<0.001). Compared to the control group, 2 μM ligustrazine treatments significantly reduced the protein levels of TLR4 and p-Akt in STR cells (all P<0.001). However, 2 μM ligustrazine treatments did not change the protein expression of Akt (P>0.05). Compared to the control group, the level of TLR4 in STR cells transfected with TLR4-mimic was significantly increased (P<0.001). Compared to the control group, transfection of TLR4-mimic reversed the anti-proliferative and pro-apoptotic effects of ligustrazine on STR cells (all P<0.001).

Conclusion: The analgesic effect of Ligustrazine on neuropathic pain caused by spinal cord injury may be related to its inhibition of the release of excitatory amino acid transmitters Glu and Gly through the TLR4/NF-κB pathway, regulation of the dynamic balance of excitatory and inhibitory amino acid neurotransmitters, and alleviation of the central sensitization effect caused by the excitotoxicity of Glu.