| Objective In order to explore MrgC/PKCε mechanism of EA analgesia,establish chronic inflammatory pain model induced by CFA(Complete freund’s adjuvant)with MrgC(Mas-related G protein-coupled receptor C)gene knocked by siRNA(small interfering RNA),observe the effects of EA(electroacupuncture)on chronic inflammatory pain and the expression,phosphorylation and membrane translocation of PKCε(protein kinase C,PKC)in DRG(dorsal root ganglion).Methods Part 1,16 health adult male Sprague-Dawley(SD)rats were randomly divided into 2 groups:control siRNA group,MrgC siRNA group,8 rats in each group.After success of intrathecal catheterization on rats,Corresponding siRNA was injected in rats of two groups for 4d,once a day,5μg/d each.The model of chronic inflammatory pain was established by subcutaneously injected CFA(0.1ml per rat)into the right hind paw at 4th day post-administration,then two groups were administrated corresponding siRNA on alternate day and executed at the 11th day post-administration.The MPWT were measured at 5 time points of pre-intrathecal catheterization,5th pre-CFA injection(After intrathecal catheterization),3th day pre-CFA injection(pre-administration),0h post-CFA injection(4th day post-administration,1d post-CFA injection(5th day post-administration),7d post-CFA injection(11th day post-administration).The expression of MrgC mRNA in ipsilateral DRG was detected by fluorogenic quantitative PCR,and the expression of MrgC receptor and p-PKCεSer729 in ipsilateral DRG was detected by IF(Immune-Fluorescence,IF).Part 2,30 health adult male Sprague-Dawley(SD)rats were randomly divided into Normal group,Model group,EA+MrgC siRNA group,EA+control siRNA group and BAM(8-22)(bovine adrenal medulla 8-22 group,BAM(8-22)group)groups,6 rats in each group.The model of chronic inflammatory pain was established by subcutaneously injected CFA(0.1ml per rat)into the right hind paw After success of intrathecal catheterization on rats.into the right hind paw Rats except Normal group(only spinal cord intrathecal catheterization).Then daily intrathecal administration,Model group,EA+MrgC siRNA group,EA+Control siRNA group were given appropriate medication intrathecal injection for 6 days,1/d,5μg/d/each,BAM(8-22)group was given the appropriate drug injection,in 1st day and 6th day,1/d,5μg/d/each,EA+MrgC siRNA group and EA+control siRNA group were given EA treatment after 30min of drug injection,and Normal group received no treatment.The HPWL(Heat Paw Withdrawal Latency,HPWL)were measured at 9 time points of CFA rats:before spinal cord intrathecal catheterization,after spinal cord intrathecal catheterization and post-CFA injection of 0,1,2,3,4,5,6 d.Use Western blot detection PKCε in the ipsilateral L4-L6 DRG and used IF detection of p-PKCε Ser729 positive expression cells at ipsilateral L4-L6 DRG.Results Part 1,Compared with 0h post-CFA injection,MPWTs of both two groups 1th day post-CFA injection decreased significantly(P<0.01).Compared with control siRNA group,the expression of MrgC mRNA of L4-L6 ipsilateral DRG each in MrgC siRNA group decreased significantly(P<0.01);The rate of MrgC receptor positive cells of L4-L6 ipsilateral DRG in MrgC siRNA group decreased significantly(P<0.01),whereas the rate of p-PKCε Ser729 positive cells increased obviously(P<0.05)at 7d post-CFA injection.Part 2:Compared with Normal group,HPWL of Model groups on 1d and 0h post-CFA injection decreased significantly(P<0.01);Compared with Model group on 6d post-CFA injection,HPWL of EA+control siRNA group was significantly increased(P<0.01);Compared with EA+control siRNA group on 6d post-CFA injection,EA+MrgC siRNA group decreased(P<0.05).Compared with Normal group,the expression of p-PKCε Ser729 of L4-L6 ipsilateral DRG in Model group increased significantly(P<0.01);Compared with Model group,the expression of p-PKCε Ser729 of L4-L6 ipsilateral DRG in EA+control siRNA group and BAM(8-22)group decreased significantly(P<0.01);Compared with EA+control siRNA group,the expression of p-PKCε Ser729 of L4-L6 ipsilateral DRG in EA+MrgC siRNA group and BAM(8-22)group increased(P<0.05).Compared with Normal group,the expression of PKCε total in Model group increased(P<0.05);Compared with EA+control siRNA group,the expression of PKCεin total cellular protein in EA+MrgC siRNA group was no statistically significant difference(P>0.05);Compared with EA+control siRNA group,the expression of PKCε menbrance/cytokeratin in EA+MrgC siRNA group was no statistically significant difference(P>0.05).Conclusion MrgC siRNA can effectively interfere the expression of MrgC mRNA and MrgC protein in L4-L6 ipsilateral DRG of rats with chronic inflammatory pain induced by CFA,and the effect of interference on MrgC receptor can obviously up-regulate the level of phosphorylation of PKCε Ser729,while it has no significant effect on MPWTs of rats.EA has a good analgesic effect on adjuvant induced chronic inflammatory pain,and possibly through up-regulation of DRG MrgC and down-regulation of phosphorylation of PKCε Ser 729. |
PDF Full Text Request