Botulinum Toxin Type A Involves Neuropathic Pain By Regulating The Expression Of P2X4R In The Spinal Cord Of Rats

Objective: To evaluate the analgesic effect of intrathecal injection of botulinum toxin type A(BoNT/A)in neuropathic pain(NP),and to explore the relationship between the analgesic effect of BoNT / A and Purine receptor P2X4(P2X4R)in the spinal cord.Methods:(1)A rat model of chronic constriction injury(CCI)of the sciatic nerve of the right hind foot was constructed.Von Frey filament pain meter was used to measure the paw withdrawal threshold(PWT)of the right hind paw of rats at 3 days before CCI modeling(d),the day of modeling(0 d),and 3 d,5 d,7 d,9 d,11 d,12 d,13 d and 14 d after modeling.The analgesic effect of intrathecal injection of 0.1 U BoNT/A was evaluated.On the 14 th day,the spinal cord tissues of Sham group,CCI model group and BoNT/A rats after intrathecal injection of CCI model were taken out.The expression of P2X4R and synaptosome-associated protein-25(SNAP-25)in spinal cord tissue was detected by Western blotting and reverse transcription-polymerase chain reaction(RT-PCR).The expression of P2X4R and SNAP-25 in the spinal cord was detected by immunofluorescence.(2)In order to further explore the relationship between the analgesic effect of intrathecal injection of BoNT/A and the inhibition of P2X4R expression in spinal cord.We evaluated the analgesic effect of IVM on BoNT / A by intraperitoneal injection of P2X4R specific activator Ivermectin(IVM).Western blotting and RT-PCR were used to detect the expression of P2X4R and SNAP-25 in spinal cord tissue of CCI model rats with intrathecal injection of BoNT / A and CCI model rats with intraperitoneal injection of IVM after intrathecal injection of BoNT/A;the expression site of P2X4R was detected by immunofluorescence co-localization.Results:(1)Our results showed that the mechanical PWT value of the right hind foot of the CCI model rats decreased significantly from the 3rd day(P<0.001),and decreased to the lowest on the 7th day and fluctuated in a small range,indicating that the CCI model was successfully constructed.After intrathecal injection of BoNT/A,the mechanical PWT value of the right hind paw was significantly increased on the 9th day(P<0.001)and remained stable until the 14 th day.Western blotting and RT-PCR results showed that the protein expression level(P<0.01)and m RNA expression level(P<0.05)of P2X4R in spinal cord tissue of CCI model rats were increased.SNAP-25 protein expression level(P<0.05)and m RNA expression level(P<0.05)increased.After intrathecal injection of BoNT/A,the expression of P2X4R and SNAP-25 was inhibited.Immunofluorescence co-localization results showed that P2X4R and SNAP-25 were mainly up-regulated in the ipsilateral spinal dorsal horn of CCI model rats,and BoNT/A could inhibit the up-regulation of P2X4R and SNAP-25 in the ipsilateral spinal dorsal horn.(2)The results of our study showed that the mechanical PWT value of the right hind foot of the CCI model rats with intrathecal injection of BoNT/A followed by intraperitoneal injection of 2 mg/Kg IVM on the 9th day was significantly lower than that of the rats injected with BoNT/A alone(P<0.001).The results of Western blotting and RT-PCR showed that the expression levels of P2X4R protein(P<0.05)and m RNA(P<0.001)in spinal cord tissue of CCI model rats injected with 2 mg / Kg IVM again after intrathecal injection of BoNT/A were higher than those of CCI model rats injected with BoNT/A only.The protein expression level(P<0.01)and m RNA expression level(P<0.001)of SNAP-25 increased again.Immunofluorescence results showed that the expression of P2X4R in the spinal dorsal horn increased again after intraperitoneal injection of IVM.Conclusion: Intrathecal injection of 0.1 U BoNT / A exerted a good analgesic effect in the rat CCI model.Intrathecal injection of 0.1 U BoNT / A produced analgesic effect in CCI model rats by inhibiting the expression of P2X4R in spinal cord.