Study On The Mediating Effect Of Oxymatrine On N- And L-type Calcium Channels In Neuropathic Pain Analgesia

Purpose:To investigate the improvement and adjustment of oxymatrine?OMT?on the gait behavior of rats and mice with neuropathic pain and to clarify N-and L-type calcium channels in the analgesic effect of OMT on neuropathic pain.Material and method:1.The neuropathic pain models were established through carrying partial sciatic nerve ligation?PSNL?on mice and carrying spinal nerve ligation?SNL?on rats.2.CatWalk analysis system was used to collect and analyze the natural gait index of mice with neuropathic pain,and the time course responses of gait behavior parameters were evaluated.The parameters include:SFI,Print Length?cm?,Print Width?cm?,Print Area?cm²?,Max Contact Area?cm²?,Stands?s?,Single Stance?s?,Max Contact Max Intensity,Mean Intensity,Regularity Index?%?,Swing?s?,Stride Length?cm?,Body Speed?cm/s?.3.The natural gait of neuropathic pain rats and mice were collected by CatWalk analysis system.The changes of gait behavior in sham operated,PSNL,OMT+PSNL group were analyzed,and the effects of OMT on this change were estimated.4.With Fluo-3 AM fluorescent probing,the intracellular calcium ion concentration was detected in primary cultured DRG neurons from the following groups:control,Sham,OMT,Nif,CTX,OMT+Nif,OMT+CTX,L-Type CC Agonist,N-Type CC Agonist,L-Type CC Agonist+OMT,N-Type CC Agonist+OMT,PSNL,OMT+PSNL,OMT+PSNL,Nif+PSNL,CTX+PSNL,Nif+OMT+PSNL,CTX+OMT+PSNL.The effects of OMT and N-,L-type calcium channel blockers were compared on the intracellular calcium ion concentration in DRG neurons of PSNL mice and the changes of intracellular calcium ion concentration in DRG neurons of normal mice with N-,L-type calcium channel agonist were compared.5.The N-,L-calcium channel-mediated OMT effects on the expression of neurotransmitter/neuropeptide and cytokines of mice were investigated by ELISA method.6.The natural gait indexes of neuropathic pain mice were collected by CatWalk analysis system,and the changes of gait behavior of PSNL mice in OMT+CTX,OMT+Nif were analyzed,then the similar effects of OMT and CTX,Nif on these CatWalk parameters were compared.Results:1.Time course response of gait behavior in neuropathic pain miceThe sciatic nerve function index?SFI?of PSNL mice was dramatically decreased at PSNL Day1?3?7?14?21?P<0.05?.On the left hindpaw?LH?of PSNL mice at each time point,compared with Day 0,the four parameters related to footprints:Print Length?cm?,Print Width?cm?,Print Area?cm²?,Max Contact Area?cm²?and the four parameters related to balance?Stands?s?,Single Stance?s?,Max Contact Max Intensity,Mean Intensity?were dramatically decreased?P<0.05?,and there was no significantly difference among the four parameters related to body coordination?P>0.05?.2.The influence of OMT on the gait behavior of neuropathic pain mice by CatWalk gait analysisUsing PSNL Day7 mice as PSNL group,compared with PSNL group,the SFI in OMT group was significantly increased?P<0.01?;On the LH,among the four parameters related to footprints,the parameters of OMT group were significantly increased than those of PSNL group?P<0.05?,and there was no significant difference among the four parameters related to body coordination?P>0.05?.The changes of four parameters associated with footprints on the right front paw?RF?and those of LH were the opposite,and there was statistical significant?P<0.05?.3.The influence of OMT on the gait behavior of neuropathic pain rats by CatWalk gait analysisThe SFI was dramatically decreased in SNL group?P<0.01?and significantly increased in OMT group?P<0.01?;the four parameters related to footprints and balance were dramatically decreased on the LH of SNL rats?P<0.01?,and the four parameters related to balance of OMT group were significantly increased than those of SNL group?P<0.05?,but there was no significant difference among the four parameters related to body coordination?P>0.05?.4.Involvement of N-,L-calcium channel in OMT-affected intracellular calciumion concentration in the DRG neurons of PSNL miceComparing to the Sham operated group,the calcium ion concentration was significantly increased?P<0.01?in PSNL group.Whilest OMT,Nif,CTX,OMT+Nif and OMT+CTX administration remarkably suppressed this elevation?P<0.01?.N-Type CC Agonist and L-type CC Agonist at both low and high doses significantly increased the intracellular calcium concentration in control group?P<0.01?,which were inhibited significantly by administration of OMT?P<0.01 and P<0.05?.5.Involvement of N-,L-calcium channel in OMT-affected changes of neurotransmitter/neuropeptide content.In the brain tissue of PSNL mice,the changes of GABA and 5-HT induced by PSNL were reversed significantly by CTX and Nif alone,as well as by the combination of OMT with calcium channel blockers.At the same time,the combination of N-or L-type calcium channel blocker with OMT significantly enhanced the inhibitory effect of OMT on the changes of GABA and 5-HT levels induced by PSNL?P<0.01?P<0.05?.In the spinal cord tissue of PSNL mice,the changes of GABA and 5-HT induced by PSNL were reversed significantly by CTX and Nif alone,as well as by the combination of OMT with calcium channel blockers?P<0.05?.At the same time,the combination of N-or L-type calcium channel blocker with OMT significantly enhanced the inhibitory effect of OMT on the changes of GABA levels induced by PSNL?P<0.01?.In the DRG tissue of PSNL mice,the changes of GABA,5-HT and SP induced by PSNL were reversed significantly by CTX and Nif alone,as well as by the combination of OMT with calcium channel blockers.At the same time,the combination of OMT with N-type calcium channel blocker significantly enhanced the inhibitory effect of OMT on the changes of GABA level induced by PSNL?P<0.05?.6.Involvement of N-,L-calcium channel in OMT-affected changes of IL-1?and HIF-?content.In the brain tissue of PSNL mice,the level of IL-1?was significantly increased?P<0.01?,and the changes of IL-1?induced by PSNL were reversed significantly by CTX and Nif alone,as well as by the combination of OMT with calcium channel blockers.The level of HIF-?was not affected by the administration of drugs.In the spinal cord tissue of PSNL mice,the levels of IL-1?and HIF-?were significantly increased?P<0.05?,the changes of IL-1?induced by PSNL were reversed significantly by CTX alone and the combination of OMT with calcium channel blockers,but not by Nif alone.There is no significant effect on the level of HIF-?.In the DRG tissue of PSNL mice,the levels of IL-1?and HIF-?in PSNL group were significantly increased?P<0.01?.Neither OMT nor L-calcium channel blocker Nif alone had significant effect on the level of HIF-?.The changes of IL-1?and HIF-?induced by PSNL were reversed significantly by CTX and Nif alone,as well as by the combination of OMT with calcium channel blockers?P<0.05?.7.The influence of N-,L-calcium channel-mediated OMT effects on the gait behavior of neuropathic pain mice by CatWalk gait analysisCompared with PSNL group,the SFI in OMT+CTX group and OMT+Nif group was significantly increased?P<0.01?;On the LH,the four parameters related to footprints in OMT group,OMT+CTX group and OMT+Nif group were significantly increased than those in PSNL group?P<0.05?,meanwhile,compared with OMT+CTX group,the index of Print Area decreased more significantly in OMT+Nif group?P<0.01?.On the RF,in OMT+CTX group and OMT+Nif group,the four parameters related to footprints were dramatically decreased?P<0.05?,the four parameters related to balance were significantly increased?P<0.05?,but the four parameters related to body coordination didn't show any changes?P>0.05?.Conclusion:1.OMT is beneficial to the recovery of SFI in mice with neuropathic pain,and can improve the relative indexes of footprints and balance on the left hindpaw,and has no effect on the Regularity Index and the body coordination.The pain caused by PSNL on the left hindpaw can induce the transfer of barycenter offset to the right front paw,and OMT can take a turn and improve worse condition in PSNL LH.2.OMT inhibited the influx of Ca²⁺stimulated by N-,L-calcium channel agonist.It showed inhibition effects on the increase of intracellular calcium concentration in DRG cells of PSNL mice when the two calcium channel blockers were used alone or separately combined with OMT was used.3.The regulating effects of OMT,N-and L-calcium channel blocker on the changes of GABA and 5-HT induced by PSNL are significant in brain,spinal and DRG tissues.This effect of OMT was enhanced by the combination with N-or L-calcium channel blockers.Both OMT and N-or L-calcium channel blockers significantly inhibited the increase of SP in DRG of PSNL mice.Meanwhile,OMT regulated N-and L-calcium channel agonist induced changes of GABA and 5-HT level in DRG.Therefore,we deduced that the effects of OMT on the changes of GABA and 5-HT levels in PSNL mice may be related to its regulating N-,L-calcium channel,and so it does on the change of SP in DRG.4.OMT can inhibit the increase of IL-1?level induced by both N-and L-calcium channel agonist.OMT,N-and L-calcium channel blockers regulated the changes of IL-1?in the brain and spinal tissues from PSNL mice?except for L-calcium channel blockers in the spinal cord?,but had no effect on the change of HIF-?.These results suggest that OMT changes the level of IL-1?may be related to its blocking of N-,L-calcium channel,but the change of HIF-?is not related to N-and L-calcium channel.5.Without affecting the body coordination of PSNL mice,OMT+CTX?OMT+Nif obviously improved and corrected the gait behavior of neuropathic pain model mice,but there was no significant difference when comparing these treatments to OMT alone treatment,which proved that additional administration of N-type calcium channel blocker CTX or L-type calcium channel blocker NIF neither increased nor reduced OMT effect.These results suggested that the improvement of gait parameters of PSNL mice by OMT might be similar to that by CTX or Nif,which works by regulating N-or L-calcium channels.