| ObjectiveFirst to investigate the effect of emodin on pain-related behaviors including spontaneous pain score,mechanical withdrawl threshold(MWT)and thermal withdrawl latency(TWL)in chronic compress injury(CCI)model.Second to explore the analgesic target of emodin by detecting differently-expressed protein with the proteomic technology,further biological information analysis and the validation of these protein with westernblot and RT-PCR.Method1.The behavioral manifestations of CCI model with different degrees of silk ligature tightnessEighty male Sprague-Dawley(SD)rats were randomly divided into Sham,Loose,Middle and Tight groups,with 20 rats in each group.Rats were evaluated on general observations,MWT and TWL before and 3,7,11,15,21 days after operation.2.The effect of different dosages of emodin on the behavioral manifestations of CCI model162 male SD rats were randomly divided into sham group,CCI group,pregabalin group(15mg/kg),and high-dosage(DH),moderate-dosage(DM),low-dosage(DL)group respectively given 100 mg,50 mg and 25 mg/kg emodin once a day,fifteen days in a row.Rats were evaluated on spontaneous score,MWT and TWL before and 3,7,11,15 after operation.L4-L6 ipsilateral spinal cords were removed after behavioral tests at day 15.3.Analgesic mechanism of emodin in CCI modelThe differently-expressed protein in L4-L6 ipsilateral spinal cords was sreened among sham group,CCI group and DM group with the proteomic technology.Functional annotation of these protein was preformed including Gene Ontology,cluster analysis,kyoto gene and genome encyclopedia(KEGG)analysis.4.The validation of the differently-expressed proteinThe differently-expressed protein was tested by westernblot and RT-PCR,such as myelin protein P0(MPZ),myelin basic protein(MBP),calcium/calmodulin dependent protein kinase(CaMKII?),calcitonin(CALCA),phospholipase C? 1(PLC? 1),protein kinase(PKC),tyrosine kinase B(TrkB)and syntaxin 1A(STX1A).Result1.The behavioral manifestations of CCI model with different degrees of silk ligature tightnessAlthough rats in both the Middle and Tight groups showed persistent abnormalities of gait and posture,reduced activity was observed only in the Tight group.Compared with the Sham group,MWT and TWL in the Middle,Loose,and Tight groups were decreased on postoperative day 3(P<0.01).On day 11,MWT and TWL in the Loose group returned to preoperative levels.In the Tight group,MWT and TWL recovered gradually,and no significant difference in TWL between this and the Sham group on day 21(P>0.05).Finally,MWT and TWL declined markedly only in the Middle group,and remained low compared with those of the Sham group(P<0.01).2.The effect of different dosages of emodin on the behavioral manifestations of CCI modelOn postoperative day 3,Compared with,spontaneous pain score of the rats in CCI group,DH group,DM group,DL group and pregabalin group increased significantly,but MWT and TWL markedly decreased(P<0.01).Until day 7,compared with the CCI model,spontaneous pain score in DH group,DL group and pregabalin group reduced progressively,and MWT and TWL simultaneously rised,particularly DM and pregabalin group(P<0.01).On day 15,compared with the CCI model,spontaneous pain score declined in DL group,and MWT rised(P<0.01),but TWL not obviously.3.Analgesic mechanism of emodin in CCI modelIn L4-L6 ipsilateral spinal cords,177 differently-expressed protein was identified among sham group,CCI group and DM group with the proteomic technology(>1.2 fold,P<0.05).Compared with the sham group model,102 differently-expressed protein was identified in CCI model with 52 up-regulation and 50 down-regulation.Compared with the CCI model,109 differently-expressed protein was identified in DM group with 66 up-regulation and 43 down-regulation.Then enrichment analysis of above protein was performed on biological function and molecular function.Compared with the CCI model,the biological function of up-regulated protein in DM group was enriched in glial cell development,astrocyte differentiation,vitamin transport;molecular function was enriched in oxygen transporter activity,oxygen binding activity.And biological function of down-regulated protein in DM group was enriched in synaptic signal release,presynaptic process,and neurotransmitter secretion and transhipment;molecular function was enriched in ion channel inhibition activity,protein kinase activity,and adenine nucleotide binding.In the end,enrichment analysis of above protein was performed on KEGG.Compared with the CCI model,KEGG of the differently-expressed protein in DM group was enriched in long-term potentiation,calcium ion signal pathway,inflammatory mediator regulation of TRP channels,MAPK signal pathway and so on.4.The validation of the differently-expressed protein by western blotCompared with the sham group,CCI can induce upregulation of CAMK? CALCA,PKC,PLC? 1,TrkB and STX1A;On the contrary,emodin may reduce the expression of the above protein.The expression of MBP and MPZ in the CCI model was lower than that in the sham group;and emodin may lead to up-regulation of the two protein.5.The validation of the differently-expressed protein by RT-PCRCompared with the sham group,the mRNA expression of CALCA,TrkB,PLC?1 significantly increase in CCI model(P<0.01);the expression of PKC,CaMKII? showed no difference(P>0.01).Compared with the CCI model,the mRNA expression of the above five protein in DM group had been on a deline(P<0.05).The mRNA expression of MBP and MPZ was lower significantly than the sham group(P<0.01);Compared with the CCI model,the expression of the two protein increased in DM group(P>0.05).ConclusionFirstly,based on the exisiting literatures,this study explored the behavioral manifestations of CCI model with different degrees of silk ligature,and found that achieving a suitable degree of tightness of silk ligation for CCI model required the applicaton of a little force after encountering resistance when the first knot is tied,such that the diameter of the sciatic nerve is barely constricted.Secondly,on the basis of previous research,we further investigated the effect of different dosages of emodin on the behavioral manifestations of CCI model,and found that emodin significantly attenuated CCI induced hyperalgesia and allodynia,which was particularly obvious in DM group.Thirdly,to explore the analgesic mechanism of emodin in CCI,the differently-expressed protein was screened by the proteomic technology and verified by western blot and RT-PCR.The results found that on the one hand emodin could increase the expression of MBP and MPZ in spinal cord and promote myelin regeneration;on the other hand it inhibited PLC?-PKC pathway,reduced the expression CaMK??,TrkB,and alleviated central sensitization and neuronal excitability. |
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