| ObjectiveWe investigated the antinociceptive effect of Aconiti Radix Cocta in complete freund's adjuvant(CFA)-induced chronic inflammatory pain in mice and its possible mechanisms associated with Kappa opioid system and TRPV1 ion channel.MethodsWe investigated the antinociceptive effect of Aconiti Radix Cocta Through behavioral way about Mechanical and Thermal stimuli and study its possible mechanisms associated with Kappa opioid system and TRPV1 ion channel.For Kappa opioid,we investigated the antinociceptive effect of Aconiti Radix Cocta by using ?,?,?antagonist and anti-dynorphin A(1-13)antiserum in CFA-induced mode.Meanwhile,we evaluated the side effects of Aconiti Radix Cocta by rotarod,open-field tests.Further,involvements of TRPV1 in Aconiti Radix Cocta antinociception were evaluated in CFA-induced TRPV1-/-and TRPV1+/+ mice,and Aconiti Radix Cocta antinociception were also evaluated in capsaicin-induced mode by nociceptive responses,Mechanical and Thermal stimuli;further more,body temperature measurement were carried out to assess its side effects.in vitro,calcium imaging was performed in HEK293T-TRPV1 cells by TRPV1 plasmid transfection to evaluate fluorescence intensity after Aconiti Radix Cocta were gived.Meanwhile,Cell viability assay was carried out to the Cytotoxicity of Aconiti Radix Cocta.1.To investigate the antinociceptive effect of Aconiti Radix Cocta in CFA-induced mice.48 healthy male ICR mice were randomly divided into six groups(n=8),the control group,the CFA group,(CFA+Aconiti Radix Cocta 0.34,0.68,and 1.35 g/kg,respectively),and control mice treated with Aconiti Radix Cocta group(1.35 g/kg),CFA or control groups received an intraplantar injection of 20?l of CFA or vehicle(tween 80 plus saline)to the right hind paw.48 h later,mice were orally administrated by syringe feeding with distilled water or Aconiti Radix Cocta daily for 7 days and mechanical and thermal hypersensitivities were evaluated.2.To investigate the antinociceptive effect of Aconiti Radix Cocta in CFA-induced mice involvements of Kappa opioid system.(1)36 healthy male ICR mice were randomly divided into six groups.the control group,the CFA group;Aconiti Radix Cocta group(1.35g/kg);Aconiti Radix Cocta+?,?,and ? antagonist,respectly;antagonist groups were pretreated with?,?,or?antagonist,respectly(cyprodime,naltrindole,nor-BNI);second,animals received an administration of Aconiti Radix Cocta(1.35g/kg),control and CFA group received distilled water,and paw withdrawal threshold of Mechanical stimuli of injured paw was evaluated 1 h later.In another set of experiments,mice were randomly divided into four groups,as the control group,the CFA group;Aconiti Radix Cocta group(1.35g/kg);Aconiti Radix Cocta+nor-BNI group;mice co-treated with Aconiti Radix Cocta and nor-BNI were subjected to the plantar,Thermal stimuli test and hot plate test for paw withdrawal latency was tested,respectly.(2)24 healthy male ICR mice were randomly divided into four groups,the control group,the CFA group;Aconiti Radix Cocta group(1.35g/kg);Aconiti Radix Cocta+anti-dynorphin A(1-13)antiserum group,mice co-treated with Aconiti Radix Cocta and anti-dynorphin A(1-13)antiserum were received a dose of anti-dynorphin A(1-13)antiserum,then,Aconiti Radix Cocta(1.35g/kg)was immediately administrated,drug gived of other groups refered to above,and mechanical or heat hypersensitivities were measured 1 h later.(3)ICR mice were randomly divided into two groups,the control group,Aconiti Radix Cocta group(1.35g/kg);mice were subjected to rotarod test 1 h after Aconiti Radix Cocta(1.35g/kg)or vehicle administration.The number of falls and latency to first fall from the apparatus were recorded for duration of 4 min.To exclude possible nonspecific muscle relaxant or sedative effects,mice were subjected to the open-field test the number of squares crossed with all paws and times of leg lifted was counted in a 5-min session.3.To investigate the antinociceptive effect of Aconiti Radix Cocta in CFA-induced mice involvements of TRPV1 ion channel.(1)48 healthy male C57BL/6 TRPV1 mice(WT)and 48 healthy male C57BL/6 TRPV1-/-mice(KO)were randomly divided into six groups(n=8)respectly,the WT-control group;the WT-CFA group;the WT-Aconiti Radix Cocta group(1.35g/kg);The WT-control group,the WT-CFA group;the WT-Aconiti Radix Cocta group(1.35 g/kg);First,baseline sensory thresholds in TRPV1+/+ and TRPV1-/-mice were tested,then mice received an intraplantar injection of 20 ?l of CFA to the right hind paw.48 h later,mice were orally administrated by syringe feeding with PCW(1.35g/kg)or distilled water,paw withdrawal threshold of the injured paw was assessed before and 0.5,1,2,3,4h post-Aconiti Radix Cocta or distilled water administration,respectively.(2)30 healthy male ICR mice were randomly divided into five groups,the control group,the Capsaicin group;Capsaicin.AMG9810 group;Capsaicin.Aconiti Radix Cocta(1.35g/kg)group;Capsaicin+ Aconiti Radix Cocta(1.35g/kg)+ nor-BNIgroup.Mice were pretreated with AMG9810(a selective TRPV1 antagonist),or Nor-BNI,then Aconiti Radix Cocta(1.35g/kg)or distilled water was administrated.prior to the injection of 20 ?l capsaicin or vehicle to the plantar surface of the right hind paw,respectively.Immediately after capsaicin application,mice were placed into clear observation boxes and the nociceptive response was evaluated as the time of spent licking the injected paw during 5 min.In another set of experiments,the same grouped mice were measured for mechanical or heat hypersensitivities after capsaicin administration for 15 min.(3)TRPV1 sequence was amplified from homo genome and cloned into plasmid,and TRPV1 plasmid was transfected into HEK293T cells by lipofectamine2000,and Cells seeded on 6-well plate,and cultured in calcium-containing DMEM overnight,then treated with PCW(0.25,0.5,1?g/ml,respectively)for 30 min.Fluo-4AM(40 ?M)was loaded into HEK293T-TRPV1 cells for 30 min at 37 ? and then 30 min at room temperature.Cells were then treated with capsaicin.Fluorescence changes were examined by a confocal microscope.Results1.the antinociceptive effect of Aconiti Radix Cocta.(1)the antinociceptive effect of Aconiti Radix Cocta in CFA-induced miceCFA caused significant mechanical hypersensitivity characterized by the reduced paw withdrawal threshold compared with control group(P<0.05).Oral administration of Aconiti Radix Cocta was able to significantly reverse mechanical hypersensitivity dose dependly(P<0.05),and this effect lasted up to 3h in high dose group.The maximum effect was observed 1 h post PCW treatment.Meanwhile,Oral administration of Aconiti Radix Cocta was able to significantly increase thermal hypersensitivity dose dependly(P<0.05).Notably,on the 7th day,PCW reduced mechanical hypersensitivity with a time-course effect profile similar to that of the first day,ruling out the possibility of drug tolerance.2.To investigate the antinociceptive effect of Aconiti Radix Cocta in CFA-induced mice involvements of Kappa opioid system paw withdrawal latency paw withdrawal threshold.(1)paw withdrawal latency and paw withdrawal threshold was strikingly reduced 48h post CFA treatment.Aconiti Radix Cocta(1.35g/kg)significantly reversed paw withdrawal threshold,and applications of ?-or?-opioid receptor antagonist cyprodime or naltrindole did not alter the antinociceptive effect of Aconiti Radix Cocta.Interestingly,when nor-BNI,a selective antagonist of K-opioid receptor,was used,the antinociceptive effects of Aconiti Radix Cocta significantly reduced.Meanwhile,Aconiti Radix Cocta also significantly increased paw withdrawal latency of Thermal stimuli and hot plate stimuli compared to CFA group.And nor-BNI also significantly decreased paw withdrawal latency of Thermal stimuli and hot plate stimuli compared to Aconiti Radix Cocta group.(2)Effects of anti-dynorphin A(1-13)antiserum to the antinociceptive effect of Aconiti Radix Cocta,the paw withdrawal threshold and paw withdrawal latency of CFA group was strikingly reduced compared to control group(P<0.05).while Aconiti Radix Cocta(1.35g/kg)significantly reversed paw withdrawal threshold and paw withdrawal latency;and these effects could be significantly decreased by coadministration of anti-dynorphin A(1-13)antiserum,with similar inhibition as in the nor-BNI-treared mice described above.(3)Effects of Aconiti Radix Cocta to the locomotor activity in mice,on the rotarod tests,The number of falls and latency to first fall from the apparatus of the mice in Aconiti Radix Cocta group were similar to control group.Meanwhile,on the open-field test,the number of squares crossed with all paws and times of leg lifted of the mice in Aconiti Radix Cocta group were also similar to control group.3.To investigate the antinociceptive effect of Aconiti Radix Cocta in CFA-induced mice involvements of TRPV1 ion channel.(1)By contrast,CFA reduced the paw withdrawal latency in wild-type mice,but not in TRPV1-/-group.Therefore,paw withdrawal threshold were chosen for the assessments of TRPV1 ion channel in the antinociceptive effects of Aconiti Radix Cocta.48 h after CFA injection,both TRPV1+/+ and TRPV1-/-mice exhibited decreased paw withdrawal threshold(P<0.05).Aconiti Radix Cocta caused a rebound of mechanical sensitivity toward baselines both in TRPV1+/+ and TRPV1-/-mice 0,5,1 and 2 h post treatment,and the inhibitions in TRPV1+/+mice higher than that in TRPV1-/-group at 0.5 and 1 h post PCW treatment.These data suggested that TRPV1 relate to the antinociceptive effect of PCW.(2)To investigate the antinociceptive effect of Aconiti Radix Cocta in capsaicin-induced mice,capsaicin induced obvious spontaneous nociception(P<0.05),and the increased licking time of nociception was significantly reduced in Aconiti Radix Cocta(1.35g/kg)and AMG9810 groups(P<0.05).and this nociception was significantly reduced in AMG9810 and Aconiti Radix Cocta-treated groups with similar inhibitions of 52±3%and 54±4%,respectively.While pretreated with nor-BNI,Aconiti Radix Cocta produced an inhibition of 21±2%.Capsaicin also induced obvious mechanical and thermal hypersensitivities,which were reduced by AMG9810 and Aconiti Radix Cocta with similar inhibitions,and Aconiti Radix Cocta could partly reverse these hypersensitivities with pretreatment of nor-BNI.At the same time,Aconiti Radix Cocta(1.35g/kg)did not induced hyperthermia compared to vehicle group,whil the TRPV1 antagonist AMG9810 induced a significant increase in body temperature(P<0.05).(3)To further explore the effect of Aconiti Radix Cocta on TRPV1 activity,the intracellular calcium levels were measured in HEK293T-TRPV1 cells.capsaicin significantly increased intracellular calcium in HEK293T-TRPV1 cells(P<0.05),while PCW(0.25,0.5,1 ?g/ml)dose dependently decreased capsaicin-induced calcium influx.These results suggested that Aconiti Radix Cocta has direct inhibitory effect on TRPV1 activity.Besides,we discovered that cytotoxic effects on HEK293T cells of Aconiti Radix Cocta(0.25,0.5,1 ?g/ml)groups had no difference compared with control group ·ConclusionsAconiti Radix Cocta had antinociceptive effect in CFA-induced mice Without finding drug tolerance.this antinociceptive effect related to Kappa opioid system and TRPV1 ion channel.Surprisly,the active dose of coniti Radix Cocta neithor produced common side effects of opioid-like drugs in rodents on motor performance,nor caused body temperature hyperthermia as TRPV1 antagonists.our results provided scientific reference for further study of the mechanism research of Aconiti Radix Cocta and based the foundation for expanding the pharmaceutical in clinical. |
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