| Objective To investigate the mechanism of spinal cord dorsal horn(SCDH)protein kinase A(PKA)-transient receptor potential type 1(TRPV1)signal pathway underlying the effect of electroacupuncture with low frequency on neuropathic pain in pain early period induced by spared nerve injury(SNI)in rats,including mechanical paw withdrawal threshold(PWT)and expression of PKA,TRPV1 and phosphorylation of TRPV1(p-TRPV1),calcitonin gene-related peptide(CGRP),substance P(SP).Methods In experiment 1,thirty-two male Sprague-Dawley rats were randomly divided into normal group,sham SNI group,SNI group,EA group,n=8.The rats of SNI and EA group were exposed to SNI surgery,while sham SNI group only involved exposure of the sciatic nerve and its branches without any lesion.The ipsilateral "Zusanli"(ST36)and "Kunlun"(BL60)acupoints were chosen.2 Hz EA intervention with 1 mA,2 mA,3 mA was administered continuously for each 10 minutes once every day from day 1(D1)to day 3(D 3)after surgery.The pain threshold was determined by ipsilateral mechanical paw withdrawal threshold(PWT)on base,after SNI(before EA),D 1 after EA(D 1),D 2 after EA(D 2),D 3 after EA(D 3).The ipsilateral SCDH PKA,TRPV1,p-TRPV1,CGRP,SP protein level expression were measured by western blotting(WB),n=5.The ipsilateral SCDH PKA,CGRP,SP positive cells expression were measured by immunofluorescence(IF),n=3.In experiment 2,forty male SD rats were exposed to intrachecal catheterization(i.c.)surgery,then randomly divided into normal group,SNI group,EA group,PKA activator(db-cAMP+EA)group,TRPV1 activator(capsaicin+EA)group,n=8.The surgery of SNI was same as experiment 1.Before EA treatment,db-cAMP solution was intrathecal injected(i.t.)2 μg/sit at a volume of 10，μl by adding normal saline(NS).Capsaicin solution was i.t.75 μg/sit at a volume of 10 μl by adding NS,then both groups followed by 10 μl of NS for flushing.normal,SNI group i.t.20 μl NS.PWT test was tested as same as experiment 1on base,after i.c.,after SNI(before EA),D 1 after EA(D 1),D 3 after EA(D 3).Results(1)Compared to normal group,sham SNI,SNI,EA groups had no significant effect on base PWT.After SNI surgery,compared to normal group,sham SNI,group had no significant effect on PWT at different time points;SNI groups had significantly decreased on PWT at different time points(P<0.01).After EA treatment,compared to SNI group,PWT in EA group was significantly increased at different time points(P<0.01).(2)WB results showed compared to normal group,sham SNI group ipsilateral SCDH PKA,TRPV1,p-TRPV1,CGRP,SP protein level in D 3 had no significant changes;ipsilateral SCDH PKA,p-TRPV1,CGRP,SP protein level in D 3 all significantly increased in SNI group(P<0.01)while TRPV1 protein level had no significant change.Compared to SNI group,ipsilateral SCDH PKA,p-TRPV1,CGRP,SP protein level in EA group all significantly decreased(P<0.05),while TRPV1 protein level had no significant change in D 3.(3)IF results showed compared to normal group,ipsilateral SCDH PKA,CGRP,SP positive cells expression all significantly increased in SNI group(P<0.01);compared to SNI group,ipsilateral SCDH PKA,CGRP,SP positive cells expression in EA group all significantly decreased(P<0.01)in D 3.(4)Block study via PKA activator(db-cAMP)and TRPV1 activator(capsaicin)intrathecal injected both significantly decreased ipsilateral PWT in db-cAMP+EA group and capsaicin+EA group,compared to EA group.Conclusions SCDH PKA,p-TRPV1 and pain hypersensitivity related neurotransmitters CGRP,SP up-regulation may contribution to SNI-induced mechanical allodynia.Electroacupuncture with low frequency can effectively relieve neuropathic pain,which may be related to its down-regulation of SCDH PKA-TRPV1 signal pathway and CGRP,SP. |
PDF Full Text Request