| ObjectivesTo verify the analgesic effects of the water extracts of paeoniae alba Radix(PR)and radix paeoniae rubra(RR)on peripheral nerve injury induced neuropathic pain.To investigate the suppressive effects of the water extracts of PR and RR on the activation of spinal glial cells(astrocytes and microglia)in neuropathic pain state.To verify that whether paeoniflorin(PF)and albiflorin(AF)has the analgesic effect on neuropathic pain,and observe the effect of PF and AF on activation of spinal cord dorsal horn glial cells on different time points.To further investigate the underlying mechanism of PF and AF on neuropathic pain,which reveals the material basis for the analgesic effect of PR and RR.Methods1.SD rats were subjected to chronic constriction injury(CCI)surgery,sham surgery was performed by exposing the right sciatic nerve without ligation.Rats were randomly divided into 5 groups:sham group,CCI group,CCI+PR group(2g/kg),CCI+RR group(2g/kg)and CCI+GP(gabapentin,100 mg/kg)group.Drugs were administered once a day for 14 days,starting on the second day after CCI.The values of every rat’s PWT and PWL on-1,3,7,and 15 postoperative were recorded.2.CCI rats were given water extracts of PR and RR treatment for 14 days after CCI surgrey.Rats were anesthetized on 15 days after CCI.The spinal L4-L5 segment were removed to prepare paraffin sections,which were stained with antibodies specific to markers of microglia(Iba-1)and astrocytes(GFAP),and the activation of astrocytes and microglia in spinal cord dorsal horn ipsilateral to the nerve injury were monitored.3.After CCI surgery,rats were divided into Sham group,CCI group,PF 80mg/kg group,AF 80mg/kg group.Animals in each group were given corresponding medicines for 14 days.Rats were anesthetized on 15 days after testing the pain behavior,and spinal L4-L5 segment were removed to prepare paraffin sections.The values of every rat’s PWT and PWL on-1,3,7,11 and 15 d postoperative were recorded and the activation of astrocytes and microglia in spinal cord dorsal horn ipsilateral to the nerve injury were monitored on 11 and 15 d after CCI by immunofluorescence.4.The model rats were divided into Sham group,CCI group,PF 80mg/kg group,AF 80mg/kg group.Animals in each group were given corresponding medicines for 14 days.Rats were anesthetized on 15 days after testing the pain behaviors,and spinal L4-L5 segment ipsilateral to the nerve injury were removed for analysis.The expression of p-p38,p-JNK,CX3CR1 proteins in spinal L4-L5 were observed and located by immunofluorescence and double-immunofluorescent labeling.The spinal proteins of p-p38,p-JNK,CX3CR1 were further quantified by Western Blot.The contents of cytokines(IL-1β,IL-6,TNF-α and CXCL1)in spinal L4-L5 were analyzed by ELISA.Results1.CCI could induce persistent mechanical allodynia and thermal hyperalgesia in rats starting from 3 d after surgery,which showed PWT and PWL decreased markedly(p<0.001,p<0.001).The administration of the water extracts of PR,RR and GP for 14 days increased both PWL and PWT strikingly(p<0.05,p<0.05,p<0.01),which demonstrated that PR and RR exerted remarkable analgesic effects on peripheral nerve injury induced neuropathic pain.2.CCI rats showed increased activation of spinal microglia(Iba-1)and astrocytes(GFAP)at 15 d after CCI(p<0.05,p<0.05).Both the water extracts of PR and RR had the effect on suppressing the activation of microglia(p<0.01,p<0.05),but not astrocytes(p>0.05,p>0.05).3.After administration with PF,both PWL and PWT were increased strikingly in rats after surgery.However,AF treatment significantly increased the PWT after surgery,but AF was useless against the decreased PWL induced by CCI.Activation of microglia was decreased by administration of both PF and AF at the two time points(p<0.05,p<0.05).Meanwhile,astrocytes(GFAP)were activated in CCI rats(p<0.05),and such induction was greatly reduced by AF treatment for 14 days(p<0.05),but not PF treatment(p>0.05).4.The levels of IL-1β,TNF-α and CXCL1 were markedly increased in the spinal cord of rats at day 15 after CCI.Moreover,CCI increased the expression of CX3CR1,p-p38 and p-JNK in spinal cord.The PF and AF treatment significantly decreased the IL-1β and TNF-αlevel(PF:p<0.01,p<0.05;AF:p<0.05,p<0.05),and decreased the expression of CX3CR1 and p-p38 in spinal cord(PF:p<0.01,p<0.01;AF:p<0.01,p<0.01),At the same time point,AF-treated,but not PF,showed significant effect on elevated level of p-JNK and CXCL1(p<0.05,p<0.01).Double immunostaining results indicated that that CX3CR1 and p-p38 were mainly co-localized in spinal cord microglia,while p-JNK was mainly co-localized in astrocytes.Conclusions1.The administration of PR&RR and PF&AF exerted remarkable analgesic effects on CCI-induced neuropathic pain.PF and AF both inhibited the activation of microglia.Moreover,AF further displayed remarkable effects on inhibiting the activation of astrocytes.2.PF and AF both inhibited the activation of microglia.The mechanisms might be down-regulating the expression of CX3CR1,which located on the surface of microglia.Moreover,both PF and AF could inhibit the activation of p38 mitogen-activated protein kinase(p38MAPK)pathway in spinal microglia and decrease the up-regulated proinflammatory cytokines IL-1β and TNF-α.3.AF,but not PF,further displayed remarkable effects on inhibiting the activation of astrocytes,suppressing the over-elevated expression of p-JNK in astrocytes,and decreasing the content of chemokine CXCL1 in the spinal cord.4.Our results demonstrate that both PF and AF have significant antinociceptive effect on CCI rat via inhibiting neuroinflammation in spinal cord,meanwhile differential mechanisms may be involved in alleviating neuropathic pain of the two isomers.The findings complement the traditional medicine connotations of PR&RR on exerting "analgesic" effect and initially reveal the material basis for the analgesic effect of the two drugs,which not only provide scientific basis for clinical application of PR&RR,but also contribute to screening potential therapeutic agents for neuropathic pain. |
PDF Full Text Request