| Background: The purpose of this study was to investigate the causality betweethe antinociceptive effect of Bo NT/A and the expression of TRPV1 in the DRG in rats with adjuvant-arthritis pain as well as to discusse the antinociceptive mechanism of BoNT/A.Methods: SD rats(N=162)were used in this study,18 rats were selected as sham baseline control group,while the others were used complete Freund's adjuvant-(CFA)induced adjuvant-arthritis pain as the animal model,then the model rats were randomly divided into four groups :CFA group(36),1U(36),3U(36),10U(36)Bo NT/A group.After the arthritis model is induced 3 weeks successfully,Sham group and CFA group were treated with a 20?Lintra-articular saline injection,while 1U,3U,10 U Bo NT/A group received an intra-articular injection of Bo NT/A at 1U/20?L,3U/20?L,10U/20?L Bo NT/A respectively.Mechanical allodynia,thermal hyperalgesia,infrared thermal imaging were conducted at 1,5 days prior to induction of arthritis,3,7,14,21 days after induction of arthritis,and 1,3,5,7,14 days following Bo NT/A treatments.At the above time points,Quantification of TRPV1 protein levels and the percentage of TRPV1 positive neurons with CGRP,TRPV1 positive neurons with cl-SNAP-25 TRPV1 m RNA levels was determined by Western blot(WB),immunofluorescence,respectively.Furthermore,whether the Bo NT/A cleaved synaptosomal-associated protein of 25 k Da(cl-SNAP-25)can be detected in the DRG after intra-articular administration Bo NT/A and the changes of TRPV1 protein levels,the percentage of TRPV1 with CGRP positive cells and the TRPV1 m RNA level.HE Staining was used to measure the histopathology of the ankle joint.Results:1,Compared with the Sham group,CFA may significantly increase the mechanical allodynia,thermal hyperalgesia(P<0.001),and the overexpression of TRPV1 receptors in protein(P<0.001),the percentage of TRPV1 to CGRP-positive neurons(P<0.001)and the TRPV1 m RNA levels(P<0.01).2,Compared with the CFA group,Bo NT/A significantly increased Paw withdrawal threshold(PWT)to mechanical stimulation with a dose-dependent manner as the threshold reversal was more obvious at a dose of 3 U,10 U than 1 U on 3,5,and 14 days after intra-articular Bo NT/A.In a similar way,the Paw withdrawal latency(PWL)was also significantly reversed to thermal stimulation by the injection of Bo NT/A.In contrast,3 U and 10 U increased the PWL compared with the saline control,while there was no difference in 1 U compared with the saline control(P>0.05).The effects of one injection of Bo NT/A to the PWT and PWL lasted for at least 14 days,increasing within 3days(PWT:3 U and 10 U:P<0.01;PWL:3U P<0.05;10U: P<0.01)and keeping at a higher level from 5(P<0.001)to 14 days(P<0.001)post injection.However,there was no difference between the 3 U and 10 U groups in the PWT and PWL(P>0.05).3,Compared with the Sham group,the ankle surface temperature significantly increased from 3 days to 35 days after the CFA injection(P<0.001),while Bo NT/A injection(1 U,3 U,10 U)groups significantly decreased compared with the CFA group,increasing within 5 days(P<0.05)and lasted for at least 14 days(P<0.05),but there was no significant difference between each Bo NT/A dose groups(1 U,3 U,10 U)(P>0.05).4,Compared with the Sham group,there were no significant improvement in ankle synovial hyperplasia and cartilage damage situation in the CFA group after saline injection from 5 days to 14 days,while the ankle synovial hyperplasia and cartilage damage were significantly improved in Bo NT/A(1 U,3 U,10 U)groups for 5 days to 14 days compared with the CFA group,but there was no significant difference between each Bo NT/A(1 U,3 U,10 U)dose groups.5,The Bo NT/A cleaved synaptosomal-associated protein of 25 k Da(cl-SNAP-25)and cl-SNAP-25 colocalized with TRPV1 neuron cells were detected in the DRG using immunofluorescence after intra-articular Bo NT/A,which demonstrated that Bo NT/A could retrograde axonal transport into TRPV1 neuron cells in the DRG after intra-articular administration.6,Although Bo NT/A significantly reduced the levels of TRPV1 protein and the percentage of TRPV1 to CGRP-positive neurons,there were no significant changes in the m RNA levels of TRPV1 between CFA and Bo NT/A(1U,3U,10U)group after Bo NT/A axonal transport into the DRG with quantitative RT-PCR(P>0.05).Conclusion: These results demonstrated that BoNT/A can not only improve the ankle synovial hyperplasia and cartilage damage but also significantly reduces adjuvant-arthritis nociceptive behaviors,while the analgesic effect works in a dose-dependent manner.Although Bo NT/A significantly reduced the levels of TRPV1 protein,there were no significant changes in the m RNA levels of TRPV1.This study provides evidence to support that the mechanism of the antinociceptive effects of Bo NT/A might be mediated by reducing TRPV1 expression through inhibiting its plasma membrane trafficking. |
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