Botulinum Toxin Type A Involved In Neuropathic Pain By Regulating VNUT Expression In The Rat Spinal Cord

Objective: Neuropathic pain was a chronic disease characterized by abnormal pain,nociceptive hyperalgesia and spontaneous pain,which seriously affected the health and quality of life of patients.To date,effective treatments for neuropathic pain were still lacking.Previous studies have shown that ATP release from dorsal horn neurons in a vesicular nucleotide transporter（VNUT）-dependent manner was the underlying mechanism of neuropathic pain.In recent years,there had been particular interest in the use of botulinum toxin type A in the treatment of painful disorders,including neuropathic pain;however,the relationship between the analgesic effect of botulinum toxin type A and the VNUT remains unclear.The aim of this study was to investigate the analgesic effect of botulinum toxin type A in neuropathic pain and its interaction with VNUT.Methods: Male 8-week-old SD rats weighing 200-220 g were selected to simulate neuropathic pain by establishing a chronic constriction injury of the sciatic nerve（CCI）model,and the paw withdraw threshold（PWT）of the rats was measured using Von Frey hairs to identify the success of the model.The expression of VNUT and Synaptosomal-associated protein 25（SNAP-25）in rat spinal cord was detected by western Blot and reverse transcription-polymerase chain reaction（RT-PCR）and the expression and distribution of VNUT and SNAP-25 in the dorsal horn of the spinal cord were detected by immunofluorescence.The interaction between SNAP-25 and VNUT was continued to be investigated in PC12 cells by means of botulinum toxin type A intervention and lentiviral overexpression of SNAP-25.To further verify the involvement of VNUT in the analgesic process of botulinum toxin type A in rat spinal cord,the expression of VNUT in spinal cord was upregulated using lentivirus to observe whether it could reverse or block the analgesic effect of botulinum toxin type A.ATP levels in spinal cord were measured using an ATP assay kit.Western Blot and RTPCR were used to detect the expression of Purine receptor P2X4（P2X4R）,P38 mitogenactivated protein kinase（P38MAPK）,Phospho-p38-mitogen activated protein kinase（PP38MAPK）,Brain-derived neurotrophic factor（BDNF）and Ionized calcium binding adapter molecule 1（Iba1）in rat spinal cord to verify whether microglia P2X4R-BDNF pathway were involved in the analgesic process of botulinum toxin type A.Results: The PWT were significantly lower in rats 3 days after CCI induction and reached stability after 7 days（P<0.001）.Intrathecal injection of 0.1 U botulinum toxin type A remarkably increased the PWT in rats（P<0.05）,whereas 0.01 U botulinum toxin type A had no analgesic effect.In contrast to the sham group,CCI surgery elevated expression of VNUT and SNAP-25 in the ipsilateral spinal cord of rats（P<0.05）,and intrathecal injection of 0.1U botulinum toxin type A effectively suppressed CCI-induced expression upregulation of VNUT and SNAP-25 in the ipsilateral spinal cord of rats（P<0.05）.Immunofluorescence results showed that both SNAP-25 and VNUT were expressed in the superficial layers of the dorsal horn of the spinal cord,and they were co-localized in the dorsal horn of the spinal cord.In vitro experiments,our results showed that overexpression of SNAP-25 caused upregulation of VNUT expression in PC12 cells（P<0.01）,whereas botulinum toxin type A inhibited the expression level of VNUT in PC12 cells（P<0.05）.When we over-expressed VNUT in the spinal cord of CCI induced rats with botulinum toxin type A,we found behaviorally that overexpression of VNUT effectively reversed the antinociceptive effects of botulinum toxin type A.The results of ATP assay showed that botulinum toxin type A significantly inhibited the increase of ATP content in the spinal cord induced by CCI surgery（P<0.0001）.The results,combined with western blotting and RTPCR,showed that intrathecal injection of 0.1 U botulinum toxin type A significantly inhibited CCI-induced upregulation of P2X4 R,P38MAPK,P-P38 MAPK,BDNF and Iba1 expression in rat ipsilateral spinal cord（P<0.05）.Conclusions: Botulinum toxin type A exerts analgesic effects in CCI-induced neuropathic pain by regulating VNUT expression in the rat spinal cord,and the microglia P2X4R-BDNF pathway is also involved in the analgesic process of botulinum toxin type A.