Based On Spinal Cord Dorsal Horn PKC/TRPV1 Pathway Of Low Frequency Electroacupuncture To Intervention Rats Maintenance Period Of Neuropathic Pain Central Sensitization Regulation Mechanism

Objective To observe analgesic effect of low frequency electroacupuncture on pain threshold of rats with neuropathic pain and the expression of protein kinase C(PKC),transient receptor potential vanilloid type1(TRPV1),phosphorylation TRPV1(p-TRPV1),substance P(SP),calcitonin gene-related peptide(CGRP)in ipsilateral spinal cord dorsal horn of rats,it is attempted to explore the analgesic mechanism of low frequency electroacupuncture central sensitization based on spinal cord dorsal horn PKC/TRPV1 signal pathway.Methods The first part:neuropathic pain rat model was established by spared nerve injury(SNI)on healthy male SD rats.The rats were randomly divided into normal group,sham SNI group,SNI group and SNI+EA group.In our study,EA(continues wave,2Hz,1 mA,2 mA,3 mA all for 10 min,totally 30 min)was administered at ipsilateral points Zusanli(ST36)and Kunlun(BL60)for 30 min,once every day from D1 to D7(maintenance period of 7d)and D1 to D14(maintenance period of 14d)after SNI surgery.Paw withdrawal thresholds(PWTs)before SNI,after SNI and at D1,D3,D7 after EA treatment for maintenance period of 7d and before SNI,after SNI and at D1,D3,D7,D14 after EA treatment for maintenance period of 14d were tested by using Dynamic Plantar Aesthesiometer after EA were measured.Western blotting study for PKC,TRPV1,p-TRPV1,SP,CGRP expression level and immunofluorescence study for PKC,TRPV1,SP,CGRP positive cells in ipsilateral SCDH were carried out.The second part:the intrathecal catheter surgery succeeded rats were randomly divided into normal group,SNI group,EA group,PMA+EA group and capsaicin+EA group.Rats in normal group,SNI group and EA group were injected intrathecally in 20 μl NS;rats in PMA+EA group were injected in 0.5 p,g PMA(diluted by 10 μl NS)per rat and rats in capsaicin+EA group were injected in 75μg capsaicin(diluted by 10 μl NS)per rat,then respectively injected in 10 μl NS before EA treatment every time.EA treatment is the same with the first part,once a day,for totally 7 times.PWTs before intrathecal catheter surgery,after intrathecal catheter surgery,after SNI surgery 1d,and at D1,D3,D7 after EA treatment were measured respectively by using a plantar tester.Immunofluorescence study for TRPV1,SP,CGRP positive cells in ipsilateral SCDH was carried out.Results①Compared with normal group,PWTs in sham SNI group had no obvious change(P>0.05),in SNI group reduced obviously at the different time points(P<0.01);compared with SNI group,in 2Hz EA group were significantly increased at the different time points after EA treatment(P<0.05 or P<0.01).②Western blott-ing results show that,PKC,TRPV1,SP,CGRP protein level in ipsilateral SCDH of rats in SNI group were significantly increased as compared to that in normal group(P<0.05 or P<0.01);compared with SNI group,PKC,TRPV1,SP,CGRP in 2Hz EA group were significantly decreased(P<0.05 or P<0.01);the p-TRPVl had no signi-ficant difference(P>0.05).③Immunofluorescence results show that,PKC,TRPV1,SP,CGRP positive cells expressions in ipsilateral SCDH of rats in SNI group were significantly increased as compared to that in normal group(P<0.01);compared with SNI group,PKC,TRPV1,SP,CGRP in 2Hz EA group were significantly decreased(P<0.01).④Results of PKC/TRPV1 pathway agonists to EA intervention effect show that,compared with normal group,PWTs in other groups all reduced obviously at the different time points after SNI surgery(P<0.01);compared with SNI group,in EA group increased obviously(P<0.01);compared with EA group,in PMA+EA group and capsaicin+EA group reduced obviously at the different time points after EA treatment(P<0.01).Immunofluorescence results show that,compared with normal group,TRPV1,SP,CGRP positive cells expressions in ipsilateral SCDH of rats in other groups were significantly up-regulated(P<0.01);compared with SNI group,TRPV1,SP,CGRP positive cells expressions in EA group were significantly down-regulated(P<0.01);compared with EA group,TRPV1,SP,CGRP positive cells expressions in PMA+EA group were significantly up-regulated(P<0.01).Conclusions In conclusion,our experiments indicated that maintenance of neuropathic pain induced by spared nerve injury was related to up-regulation of PKC,TRPV1,SP,CGRP in ipsilateral SCDH,regardless of p-TRPV.Electroacupuncture with low frequency could alleviate neuropathic pain during the maintenance period,which may be related to the down-regulation of TRPV1 signal transduction pathway which mediated by PKC,so as SP and CGRP.